Genomic insights into host and parasite interactions during intracellular infection by Toxoplasma gondii.

To gain insights into the molecular interactions of an intracellular pathogen and its host cell, we studied the gene expression and chromatin states of human fibroblasts infected with the Apicomplexan parasite Toxoplasma gondii. We show a striking activation of host cell genes that regulate a number of cellular processes, some of which are protective of the host cell, others likely to be advantageous to the pathogen. The simultaneous capture of host and parasite genomic information allowed us to gain insights into the regulation of the T. gondii genome. We show how chromatin accessibility and transcriptional profiling together permit novel annotation of the parasite's genome, including more accurate mapping of known genes and the identification of new genes and cis-regulatory elements. Motif analysis reveals not only the known T. gondii AP2 transcription factor-binding site but also a previously-undiscovered candidate TATA box-containing motif at one-quarter of promoters. By inferring the transcription factor and upstream cell signaling responses involved in the host cell, we can use genomic information to gain insights into T. gondii's perturbation of host cell physiology. Our resulting model builds on previously-described human host cell signalling responses to T. gondii infection, linked to induction of specific transcription factors, some of which appear to be solely protective of the host cell, others of which appear to be co-opted by the pathogen to enhance its own survival.

A Cellular Stress Response Induced by the CRISPR-dCas9 Activation System Is Not Heritable Through Cell Divisions.

The CRISPR-Cas9 system can be modified to perform "epigenetic editing" by utilizing the catalytically inactive (dead) Cas9 (dCas9) to recruit regulatory proteins to specific genomic locations. In prior studies, epigenetic editing with multimers of the transactivator VP16 and guide RNAs (gRNAs) was found to cause adverse cellular responses. These side effects may confound studies inducing new cellular properties, especially if the cellular responses are maintained through cell divisions-an epigenetic regulatory property. Here, we show how distinct components of this CRISPR-dCas9 activation system, particularly dCas9 with untargeted gRNAs, upregulate genes associated with transcriptional stress, defense response, and regulation of cell death. Our results highlight a previously undetected acute stress response to CRISPR-dCas9 components in human cells, which is transient and not maintained through cell divisions.

High-efficiency genomic editing in Epstein-Barr virus-transformed lymphoblastoid B cells using a single-stranded donor oligonucleotide strategy.

While human lymphoblastoid cell lines represent a valuable resource for population genetic studies, they have usually been regarded as difficult for CRISPR-mediated genomic editing because of very inefficient DNA transfection and retroviral or lentiviral transduction in these cells, which becomes a substantial problem when multiple constructs need to be co-expressed. Here we describe a protocol using a single-stranded donor oligonucleotide strategy for 'scarless' editing in lymphoblastoid cells, yielding 12/60 (20%) of clones with homology-directed recombination, when rates of <5-10% are frequently typical for many other cell types. The protocol does not require the use of lentiviruses or stable transfection, permitting lymphoblastoid cell lines to be used for CRISPR-mediated genomic targeting and screening in population genetic studies.

Functional genetic variants can mediate their regulatory effects through alteration of transcription factor binding.

Functional variants in the genome are usually identified by their association with local gene expression, DNA methylation or chromatin states. DNA sequence motif analysis and chromatin immunoprecipitation studies have provided indirect support for the hypothesis that functional variants alter transcription factor binding to exert their effects. In this study, we provide direct evidence that functional variants can alter transcription factor binding. We identify a multifunctional variant within the TBC1D4 gene encoding a canonical NFκB binding site, and edited it using CRISPR-Cas9 to remove this site. We show that this editing reduces TBC1D4 expression, local chromatin accessibility and binding of the p65 component of NFκB. We then used CRISPR without genomic editing to guide p65 back to the edited locus, demonstrating that this re-targeting, occurring ~182 kb from the gene promoter, is enough to restore the function of the locus, supporting the central role of transcription factors mediating the effects of functional variants.

Detecting, quantifying, and discriminating the mechanism of mosaic chromosomal aneuploidies using MAD-seq.

Current approaches to detect and characterize mosaic chromosomal aneuploidy are limited by sensitivity, efficiency, cost, or the need to culture cells. We describe the mosaic aneuploidy detection by massively parallel sequencing (MAD-seq) capture assay and the MADSEQ analytical approach that allow low (<10%) levels of mosaicism for chromosomal aneuploidy or regional loss of heterozygosity to be detected, assigned to a meiotic or mitotic origin, and quantified as a proportion of the cells in the sample. We show results from a multi-ethnic MAD-seq (meMAD-seq) capture design that works equally well in populations of diverse racial and ethnic origins and how the MADSEQ analytical approach can be applied to exome or whole-genome sequencing data, revealing previously unrecognized aneuploidy or copy number neutral loss of heterozygosity in samples studied by the 1000 Genomes Project, cell lines from public repositories, and one of the Illumina Platinum Genomes samples. We have made the meMAD-seq capture design and MADSEQ analytical software open for unrestricted use, with the goal that they can be applied in clinical samples to allow new insights into the unrecognized prevalence of mosaic chromosomal aneuploidy in humans and its phenotypic associations.

Simultaneous Measurement of HDAC1 and HDAC6 Activity in HeLa Cells Using UHPLC-MS.

The search for new histone deacetylase (HDAC) inhibitors is of increasing interest in drug discovery. Isoform selectivity has been in the spotlight since the approval of romidepsin, a class I HDAC inhibitor for cancer therapy, and the clinical investigation of HDAC6-specific inhibitors for multiple myeloma. The present method is used to determine the inhibitory activity of test compounds on HDAC1 and HDAC6 in cells. The isoform activity is measured using the ultra-high-performance liquid chromatography - mass spectrometry (UHPLC-MS) analysis of specific substrates incubated with treated and untreated HeLa cells. The method has the advantage of reflecting the endogenous HDAC activity within the cell environment, in contrast to cell-free biochemical assays conducted on isolated isoforms. Moreover, because it is based on the quantification of synthetic substrates, the method does not require the antibody recognition of endogenous acetylated proteins. It is easily adaptable to several cell lines and an automated process. The method has already proved useful in finding HDAC6-selective compounds in neuroblasts. Representative results are shown here with the standard HDAC inhibitors trichostatin A (non-specific), MS275 (HDAC1-specific), and tubastatin A (HDAC6-specific) using HeLa cells.

Synthesis of an Uncharged Tetra-cyclopeptide Acting as a Transmembrane Carrier: Enhanced Cellular and Nuclear Uptake.

A small uncharged cyclopeptide scaffold inspired by a natural product and designed to undergo postfunctionalizations was used as a new transmembrane vector. A bioactive and fluorescent triazole aminocoumarin was bound to this carrier to facilitate its moving across cell and subcellular membranes, and this led to an increase in its cell toxicity.

Novel histone deacetylase 6 (HDAC6) selective inhibitors: a patent evaluation (WO2014181137).

INTRODUCTION: Histone deacetylases (HDACs) are known to deacetylate histones and other proteins, which makes HDAC inhibitors able to affect cell survival, cell signaling, transport, and gene expression. Those effects have been associated to the therapeutic success of HDAC inhibitors. Class I-selective or pan-HDAC inhibitors have been approved for cancer therapy by the US Food and Drug Administration (FDA). Moreover, HDAC6 selective inhibitors entered phase I and II clinical trials for treating multiple myeloma. The development of potent and selective HDAC inhibitors is a hot topic in current drug discovery. Areas covered: The invention described in this patent (WO2014181137) is related to hydroxamic acid derivatives with inhibitory activity towards HDACs, their synthetic process and pharmaceutical formulations, as well as a method for treating patients suffering from a list of selected tumoral, inflammatory, cardiac and chronic disorders. Expert opinion: The compounds disclosed within this patent are selective against HDAC6 and their structure is related to tubastatin A, a known HDAC6 selective inhibitor. They are newly synthesized diarylamines showing an improved selectivity profile compared to other diarylamines under clinical investigation.

UHPLC-MS-based HDAC Assay Applied to Bio-guided Microfractionation of Fungal Extracts.

INTRODUCTION: Histone deacetylases (HDAC) are considered as promising targets for cancer treatment. Today, four HDAC inhibitors, vorinostat, romidepsin, belinostat, and panobinostat, have been approved by the Food and Drug Administration (FDA) for cancer treatment, while others are in clinical trials. Among them, several are naturally occurring fungal metabolites. OBJECTIVE: To develop and optimise an enzyme assay for bio-guided identification of HDAC inhibitors in fungal strains. METHODS: Fluorescence and MS-based HDAC enzymatic assays were compared during the bio-guided fractionation of Penicillium griseofulvum. The MS-based approach was then optimised to evaluate HDAC selectivity using the human recombinant class I isoform HDAC1 and the class II isoform HDAC6. RESULTS: Fluorescence-based assays have several drawbacks when used for bio-guided fractionation because of the native fluorescence and the trypsin inhibitory ability of compounds present in many extracts. The MS-based method led to the isolation of gliocladride C, which is selective for HDAC1 and salirepol, which showed an HDAC6 selectivity. Their activity and presence in P. griseofulvum is described here for the first time. CONCLUSION: The UHPLC-ESI-MS/MS-based method using specific HDAC isoforms is suitable to isolate selective HDAC inhibitors by bio-guided fractionation of fungal strains. Also, it decreases potential interferences with natural products compared to the fluorescence-based assay.

Hydroxyl Ketone-Based Histone Deacetylase Inhibitors To Gain Insight into Class I HDAC Selectivity versus That of HDAC6.

Little is known about the biological and structural features that govern the isoform selectivity for class I histone deacetylases (HDACs) over HDAC6. In addition to that for known inhibitors, like benzamides, psammaplin A, and cyclodepsipeptide-derived thiols, selectivity was also observed for naturally occurring cyclopeptide HDAC inhibitors with an aliphatic flexible linker and ketonelike zinc-binding group (ZBG). The present study reports that this isoform selectivity is mainly due to the linker and ZBG, as replacement of the cyclopeptide cap region by a simple aniline retained class I HDAC isoform selectivity toward HDAC6 in enzymatic assays. The best cyclopeptide-free analogues preserved efficacy against Plasmodium falciparum and cancer cell lines. Molecular modeling provided hypotheses to explain this selectivity and suggests different behaviors of the flexible linker on HDAC1 and HDAC6 pockets, which may influence, on the basis of the strength of the ZBG, its coordination with the zinc ion.

Synthesis of a selective HDAC6 inhibitor active in neuroblasts.

In recent years, the role of HDAC6 in neurodegeneration has been partially elucidated, which led some authors to propose HDAC6 inhibitors as a therapeutic strategy to treat neurodegenerative diseases. In an effort to develop a selective HDAC6 inhibitor which can cross the blood brain barrier (BBB), a modified hydroxamate derivative (compound 3) was designed and synthetized. This compound was predicted to have potential for BBB penetration based on in silico and in vitro evaluation of passive permeability. When tested for its HDAC inhibitory activity, the IC50 value of compound 3 towards HDAC6 was in the nM range in both enzymatic and cell-based assays. Compound 3 showed a cell-based selectivity profile close to that of tubastatin A in SH-SY5Y human neuroblastoma cells, and a good BBB permeability profile.

Molecular dynamics of zinc-finger ubiquitin binding domains: a comparative study of histone deacetylase 6 and ubiquitin-specific protease 5.

HDAC6 is a unique cytoplasmic histone deacetylase characterized by two deacetylase domains, and by a zinc-finger ubiquitin binding domain (ZnF-UBP) able to recognize ubiquitin (Ub). The latter has recently been demonstrated to be involved in the progression of neurodegenerative diseases and in mediating infection by the influenza A virus. Nowadays, understanding the dynamic and energetic features of HDAC6 ZnF-UBP-Ub recognition is considered as a crucial step for the conception of HDAC6 potential modulators. In this study, the atomic, solvent-related, and thermodynamic features behind HDAC6 ZnF-UBP-Ub recognition have been analyzed through molecular dynamics simulations. The behavior was then compared to the prototypical ZnF-UBP from ubiquitin-specific protease 5 (USP5) in order to spot relevant differences useful for selective drug design. Principal component analysis highlighted flapping motions of the L2A loop which were lowered down upon Ub binding in both systems. While polar and nonpolar interactions involving Ub G75 and G76 residues were also common features stabilizing both complexes, salt bridges showed a different pattern, more significant in HDAC6 ZnF-UBP-Ub, whose energetic contribution in USP5 ZnF-UBP-Ub was compensated by the presence of a more stable bridging water molecule. Whereas molecular mechanics/Poisson-Boltzmann surface area (MM-PBSA) free energies of binding were comparable for both systems, in agreement with experiments, computational alanine scanning and free energy decomposition data revealed that HDAC6 E1141 and D1178 are potential hotspots for the design of selective HDAC6 modulators.

Aurones as histone deacetylase inhibitors: identification of key features.

In this study, a total of 22 flavonoids were tested for their HDAC inhibitory activity using fluorimetric and BRET-based assays. Four aurones were found to be active in both assays and showed IC50 values below 20 µM in the enzymatic assay. Molecular modelling revealed that the presence of hydroxyl groups was responsible for good compound orientation within the isoenzyme catalytic site and zinc chelation.

Indole alkaloids of Psychotria as multifunctional cholinesterases and monoamine oxidases inhibitors.

Thirteen Psychotria alkaloids were evaluated regarding their interactions with acetylcholinesterase (AChE), butyrylcholinesterase (BChE), and monoamine oxidases A and B (MAO-A and MAO-B), which are enzymatic targets related with neurodegenerative diseases. Two quaternary ß-carboline alkaloids, prunifoleine and 14-oxoprunifoleine, inhibited AChE, BChE and MAO-A with IC(50) values corresponding to 10 and 3.39 µM for AChE, 100 and 11 µM for BChE, and 7.41 and 6.92 µM for MAO-A, respectively. Both compounds seem to behave as noncompetitive AChE inhibitors and time-dependent MAO-A inhibitors. In addition, the monoterpene indole alkaloids (MIAs) angustine, vallesiachotamine lactone, E-vallesiachotamine and Z-vallesiachotamine inhibited BChE and MAO-A with IC(50) values ranging from 3.47 to 14 µM for BChE inhibition and from 0.85 to 2.14 µM for MAO-A inhibition. Among the tested MIAs, angustine is able to inhibit MAO-A in a reversible and competitive way while the three vallesiachotamine-like alkaloids display a time-dependent inhibition on this target. Docking calculations were performed in order to understand the binding mode between the most active ligands and the selected targets. Taken together, our findings established molecular details of AChE, BChE and MAO-A inhibition by quaternary ß-carboline alkaloids and MIAs from Psychotria, suggesting these secondary metabolites are scaffolds for the development of multifunctional compounds against neurodegeneration.

Ellagic acid derivatives from Syzygium cumini stem bark: investigation of their antiplasmodial activity.

Bioguided fractionation of Syzygium cumini (Myrtaceae) bark decoction for antiplasmodial activity was performed, leading to the isolation of three known ellagic acid derivatives (ellagic acid, ellagic acid 4-O-alpha-L-2"-acetylrhamnopyranoside, 3-O-methylellagic acid 3'-O-alpha-L-rhamnopyranoside), as well as the new derivative 3-O-methylellagic acid 3'-O-beta-D-glucopyranoside. Activity investigation was based on the reduction of P. falciparum (PfK1) parasitaemia in vitro and the inhibition of beta-hematin formation, a known mechanism of action of some antimalarial drugs. Among the investigated ellagic acid derivatives, only ellagic acid was able to reduce P. falciparum parasitaemia in vitro and inhibit beta-hematin formation, suggesting that free hydroxyl groups are necessary for activity within this class of compounds.

A TLC bioautographic method for the detection of alpha- and beta-glucosidase inhibitors in plant extracts.

INTRODUCTION: Bioautographic assays using TLC play an important role in the search for active compounds from plants. A TLC assay has previously been established for the detection of beta-glucosidase inhibitors but not for alpha-glucosidase. Nonetheless, alpha-glucosidase inhibition is an important target for therapeutic agents against of type 2 diabetes and anti-viral infections. OBJECTIVE: To develop a TLC bioautographic method to detect alpha- and beta-glucosidase inhibitors in plant extracts. METHODOLOGY: The enzymes alpha- and beta-d-glucosidase were dissolved in sodium acetate buffer. After migration of the samples, the TLC plate was sprayed with enzyme solution and incubated at room temperature for 60 min in the case of alpha-d-glucosidase, and 37 degrees C for 20 min in the case of beta-d-glucosidase. For detection of the active enzyme, solutions of 2-naphthyl-alpha-D-glucopyranoside or 2-naphthyl-beta-D-glucopyranoside and Fast Blue Salt were mixed at a ratio of 1 : 1 (for alpha-d-glucosidase) or 1 : 4 (for beta-d-glucosidase) and sprayed onto the plate to give a purple background colouration after 2-5 min. RESULTS: Enzyme inhibitors were visualised as white spots on the TLC plates. Conduritol B epoxide inhibited alpha-d-glucosidase and beta-d-glucosidase down to 0.1 microg. Methanol extracts of Tussilago farfara and Urtica dioica after migration on TLC gave enzymatic inhibition when applied in amounts of 100 microg for alpha-glucosidase and 50 microg for beta-glucosidase. CONCLUSION: The screening test was able to detect inhibition of alpha- and beta-glucosidases by pure reference substances and by compounds present in complex matrices, such as plant extracts.

Isolation and on-line identification of antioxidant compounds from three Baccharis species by HPLC-UV-MS/MS with post-column derivatisation.

The aqueous extracts of aerial parts of Baccharis trimera (Less.) DC., B. crispa Spreng. and B. usterii Heering (Asteraceae) displayed significant radical scavenging activity in a diphenylpicrylhydrazole (DPPH)/TLC assay. In order to rapidly identify the active principles, the crude extracts were analysed by HPLC-UV, and an HPLC-micro-fractionation of the extract of B. usterii was performed. Six quinic acids derivatives (1-6) were isolated from B. usterii by MPLC. The fractions were monitored by DPPH/TLC assay and a series of radical-scavenging quinic acid derivatives could be identified. The comparison of the HPLC profiles of the extracts of B. usterii, B. trimera and B. crispa was performed. In order to obtain complementary on-line structural information for all peaks of interest, HPLC-MS/MS together with HPLC-UV involving post-column addition of UV shift reagents was undertaken on the crude extract. The interpretation of these data permitted the on-line identification of known compounds, some of which are reported for the first time in this plant.