Upregulation of S100A8 in peripheral blood mononuclear cells from patients with depression treated with SSRIs: a pilot study.

BACKGROUND: Major depressive disorder (MDD) affects more than 350 million people worldwide, and there is currently no laboratory test to diagnose it. This pilot study aimed to identify potential biomarkers in peripheral blood mononuclear cells (PBMCs) from MDD patients. METHODS: We used tandem mass tagging coupled to synchronous precursor selection (mass spectrometry) to obtain the differential proteomic profile from a pool of PBMCs from MDD patients and healthy subjects, and quantitative PCR to assess gene expression of differentially expressed proteins (DEPs) of our interest. RESULTS: We identified 247 proteins, of which 133 had a fold change ≥ 2.0 compared to healthy volunteers. Using pathway enrichment analysis, we found that some processes, such as platelet degranulation, coagulation, and the inflammatory response, are perturbed in MDD patients. The gene-disease association analysis showed that molecular alterations in PBMCs from MDD patients are associated with cerebral ischemia, vascular disease, thrombosis, acute coronary syndrome, and myocardial ischemia, in addition to other conditions such as inflammation and diabetic retinopathy. CONCLUSIONS: We confirmed by qRT-PCR that S100A8 is upregulated in PBMCs from MDD patients and thus could be an emerging biomarker of this disorder. This report lays the groundwork for future studies in a broader and more diverse population and contributes to a deeper characterization of MDD.

The Antimalarial Drug Pyronaridine Inhibits Topoisomerase II in Breast Cancer Cells and Hinders Tumor Progression In Vivo.

BACKGROUND: Breast cancer is the most frequently diagnosed cancer in women worldwide. Pyronaridine (PND), an antimalarial drug, was shown to exert anticancer activity on seventeen different human cancer cells, seven from female breast tissue. Additionally, PND induced apoptosis via mitochondrial depolarization, alteration of cell cycle progression, and DNA intercalation. However, the molecular target of PND in cells was not elucidated. OBJECTIVE: Here, we have further investigated PND's mode of action by using transcriptome analysis. Preclinical studies were also performed to determine whether PND could affect tumor progression in a human breast cancer xenograft in mice. Moreover, we assessed the combined efficacy of PND with well-known anticancer drugs. METHODS: Transcriptome analyses of PND-treated cancer cells were performed. Topoisomerase II activity was evaluated by an in vitro assay. In addition, daily oral administration of PND was given to mice with human breast cancer xenografts. The differential nuclear staining assay measured in-vitro cell toxicity. RESULTS: The transcriptome signatures suggested that PND might act as a topoisomerase II inhibitor. Thus, topoisomerase inhibition assays were performed, providing evidence that PND is a bona fide topoisomerase II inhibitor. Also, in-vivo studies suggest that PND hinders tumor progression. Besides, combination studies of PND with anticancer drugs cisplatin and gemcitabine revealed higher cytotoxicity against cancer cells than individual drug administration. CONCLUSION: The findings provide evidence that PND is a topoisomerase II inhibitor and can hinder cancer progression in an animal model, further demonstrating PND's favorable characteristics as a repurposed anticancer drug.

Phenylpropanoids Are Connected to Cell Wall Fortification and Stress Tolerance in Avocado Somatic Embryogenesis.

Somatic embryogenesis (SE) is a valuable model for understanding the mechanism of plant embryogenesis and a tool for the mass production of plants. However, establishing SE in avocado has been complicated due to the very low efficiency of embryo induction and plant regeneration. To understand the molecular foundation of the SE induction and development in avocado, we compared embryogenic (EC) and non-embryogenic (NEC) cultures of two avocado varieties using proteomic and metabolomic approaches. Although Criollo and Hass EC exhibited similarities in the proteome and metabolome profile, in general, we observed a more active phenylpropanoid pathway in EC than NEC. This pathway is associated with the tolerance of stress responses, probably through the reinforcement of the cell wall and flavonoid production. We could corroborate that particular polyphenolics compounds, including p-coumaric acid and t-ferulic acid, stimulated the production of somatic embryos in avocado. Exogen phenolic compounds were associated with the modification of the content of endogenous polyphenolic and the induction of the production of the putative auxin-a, adenosine, cellulose and 1,26-hexacosanediol-diferulate. We suggest that in EC of avocado, there is an enhanced phenylpropanoid metabolism for the production of the building blocks of lignin and flavonoid compounds having a role in cell wall reinforcement for tolerating stress response. Data are available at ProteomeXchange with the identifier PXD019705.

Drug mechanism-of-action discovery through the integration of pharmacological and CRISPR screens.

Low success rates during drug development are due, in part, to the difficulty of defining drug mechanism-of-action and molecular markers of therapeutic activity. Here, we integrated 199,219 drug sensitivity measurements for 397 unique anti-cancer drugs with genome-wide CRISPR loss-of-function screens in 484 cell lines to systematically investigate cellular drug mechanism-of-action. We observed an enrichment for positive associations between the profile of drug sensitivity and knockout of a drug's nominal target, and by leveraging protein-protein networks, we identified pathways underpinning drug sensitivity. This revealed an unappreciated positive association between mitochondrial E3 ubiquitin-protein ligase MARCH5 dependency and sensitivity to MCL1 inhibitors in breast cancer cell lines. We also estimated drug on-target and off-target activity, informing on specificity, potency and toxicity. Linking drug and gene dependency together with genomic data sets uncovered contexts in which molecular networks when perturbed mediate cancer cell loss-of-fitness and thereby provide independent and orthogonal evidence of biomarkers for drug development. This study illustrates how integrating cell line drug sensitivity with CRISPR loss-of-function screens can elucidate mechanism-of-action to advance drug development.

Pranlukast Antagonizes CD49f and Reduces Stemness in Triple-Negative Breast Cancer Cells.

INTRODUCTION: Cancer stem cells (CSCs) drive the initiation, maintenance, and therapy response of breast tumors. CD49f is expressed in breast CSCs and functions in the maintenance of stemness. Thus, blockade of CD49f is a potential therapeutic approach for targeting breast CSCs. In the present study, we aimed to repurpose drugs as CD49f antagonists. MATERIALS AND METHODS: We performed consensus molecular docking using a subdomain of CD49f that is critical for heterodimerization and a collection of pharmochemicals clinically tested. Molecular dynamics simulations were employed to further characterize drug-target binding. Using MDA-MB-231 cells, we evaluated the effects of potential CD49f antagonists on 1) cell adhesion to laminin; 2) mammosphere formation; and 3) cell viability. We analyzed the effects of the drug with better CSC-selectivity on the activation of CD49f-downstream signaling by Western blot (WB) and co-immunoprecipitation. Expressions of the stem cell markers CD44 and SOX2 were analyzed by flow cytometry and WB, respectively. Transactivation of SOX2 promoter was evaluated by luciferase reporter assays. Changes in the number of CSCs were assessed by limiting-dilution xenotransplantation. RESULTS: Pranlukast, a drug used to treat asthma, bound to CD49f in silico and inhibited the adhesion of CD49f+ MDA-MB-231 cells to laminin, indicating that it antagonizes CD49f-containing integrins. Molecular dynamics analysis showed that pranlukast binding induces conformational changes in CD49f that affect its interaction with ß1-integrin subunit and constrained the conformational dynamics of the heterodimer. Pranlukast decreased the clonogenicity of breast cancer cells on mammosphere formation assay but had no impact on the viability of bulk tumor cells. Brief exposure of MDA-MB-231 cells to pranlukast altered CD49f-dependent signaling, reducing focal adhesion kinase (FAK) and phosphatidylinositol 3-kinase (PI3K) activation. Further, pranlukast-treated cells showed decreased CD44 and SOX2 expression, SOX2 promoter transactivation, and in vivo tumorigenicity, supporting that this drug reduces the frequency of CSC. CONCLUSION: Our results support the function of pranlukast as a CD49f antagonist that reduces the CSC population in triple-negative breast cancer cells. The pharmacokinetics and toxicology of this drug have already been established, rendering a potential adjuvant therapy for breast cancer patients.

Endorsing and extending the repertory of nutraceutical and antioxidant sources in mangoes during postharvest shelf life.

Mango byproducts, such as peels, contain high levels of antioxidants and fiber and represent important sources of nutraceuticals and pharmacological products. Fruit are collected at the mature green stage then stored and ripened, undergoing several structural and molecular changes over the course of this process. However, very little is known regarding the content and nature of antioxidant compounds in peels of elite and local cultivars during postharvest shelf life (PSL). We screened the phenolic compound content of six cultivars during PSL, including elite (Kent, Tommy, and Ataulfo) and local (Manila, Manililla, and Criollo) mangoes, using a targeted metabolomics approach. We determined that Ataulfo mangoes exhibited the highest content of phenolic compounds during PSL. Untargeted metabolomics and comparative proteomics in Ataulfo and Manililla showed these cultivars to be significant sources of phenolic and lipidic compounds, with the latter cultivar also representing an interesting candidate as a new source for nutraceutical products.

ChEMBL: towards direct deposition of bioassay data.

ChEMBL is a large, open-access bioactivity database (https://www.ebi.ac.uk/chembl), previously described in the 2012, 2014 and 2017 Nucleic Acids Research Database Issues. In the last two years, several important improvements have been made to the database and are described here. These include more robust capture and representation of assay details; a new data deposition system, allowing updating of data sets and deposition of supplementary data; and a completely redesigned web interface, with enhanced search and filtering capabilities.

A recent advance in the intracellular and extracellular redox post-translational modification of proteins in plants.

Plants, as sessile organisms, have acquired through evolution sophisticated regulatory signal pathways to overcome external variable factors during each stage of the life cycle. Among these regulatory signals, two pathways in particular, reactive oxygen species and reactive nitrogen species, have become of significant interest in several aspects of plant biology, underpinning these molecules as critical regulators during development, cellular differentiation, and plant-pathogen interaction. Recently, redox posttranslational modifications (PTM), such as S-nitrosylation on cysteine residues and tyrosine nitration, have shed light on multiple protein targets, as they are associated with signal networks/downstream metabolic pathways, capable of transducing the imbalance of redox hemostasis and consequently redirecting the biochemical status under stress conditions. However, most of the redox PTM have been studied only in the intracellular compartment, providing limited information concerning redox PTM in the extracellular matrix of plant cells. Nevertheless, recent studies have indicated the plausibility of redox PTM in extracellular proteins, including cell wall associated proteins. Accordingly, in this review, we endeavor to examine evidence of redox PTM supported by mass spectrometry data in the intracellular and extracellular space in plant cells. As a further example, we focus the last section of this review on illustrating, using molecular dynamics simulation, the effect of S-nitrosylation on the structural conformation of well-known cell wall-associated proteins including pectin methylesterase and xyloglucan endo-transglycosylases.

Whole-Genome Sequencing and Comparative Genome Analysis Provided Insight into the Predatory Features and Genetic Diversity of Two Bdellovibrio Species Isolated from Soil.

Bdellovibrio spp. are predatory bacteria with great potential as antimicrobial agents. Studies have shown that members of the genus Bdellovibrio exhibit peculiar characteristics that influence their ecological adaptations. In this study, whole genomes of two different Bdellovibrio spp. designated SKB1291214 and SSB218315 isolated from soil were sequenced. The core genes shared by all the Bdellovibrio spp. considered for the pangenome analysis including the epibiotic B. exovorus were 795. The number of unique genes identified in Bdellovibrio spp. SKB1291214, SSB218315, W, and B. exovorus JJS was 1343, 113, 857, and 1572, respectively. These unique genes encode hydrolytic, chemotaxis, and transporter proteins which might be useful for predation in the Bdellovibrio strains. Furthermore, the two Bdellovibrio strains exhibited differences based on the % GC content, amino acid identity, and 16S rRNA gene sequence. The 16S rRNA gene sequence of Bdellovibrio sp. SKB1291214 shared 99% identity with that of an uncultured Bdellovibrio sp. clone 12L 106 (a pairwise distance of 0.008) and 95-97% identity (a pairwise distance of 0.043) with that of other culturable terrestrial Bdellovibrio spp., including strain SSB218315. In Bdellovibrio sp. SKB1291214, 174 bp sequence was inserted at the host interaction (hit) locus region usually attributed to prey attachment, invasion, and development of host independent Bdellovibrio phenotypes. Also, a gene equivalent to Bd0108 in B. bacteriovorus HD100 was not conserved in Bdellovibrio sp. SKB1291214. The results of this study provided information on the genetic characteristics and diversity of the genus Bdellovibrio that can contribute to their successful applications as a biocontrol agent.

A structure- and chemical genomics-based approach for repositioning of drugs against VCP/p97 ATPase.

Valosin-containing protein (VCP/p97) ATPase (a.k.a. Cdc48) is a key member of the ER-associated protein degradation (ERAD) pathway. ERAD and VCP/p97 have been implicated in a multitude of human diseases, such as neurodegenerative diseases and cancer. Inhibition of VCP/p97 induces proteotoxic ER stress and cell death in cancer cells, making it an attractive target for cancer treatment. However, no drugs exist against this protein in the market. Repositioning of drugs towards new indications is an attractive alternative to the de novo drug development due to the potential for significantly shorter time to clinical translation. Here, we employed an integrative strategy for the repositioning of drugs as novel inhibitors of the VCP/p97 ATPase. We integrated structure-based virtual screening with the chemical genomics analysis of drug molecular signatures, and identified several candidate inhibitors of VCP/p97 ATPase. Importantly, experimental validation with cell-based and in vitro ATPase assays confirmed three (ebastine, astemizole and clotrimazole) out of seven tested candidates (~40% true hit rate) as direct inhibitors of VCP/p97 and ERAD. This study introduces an effective integrative strategy for drug repositioning, and identified new drugs against the VCP/p97/ERAD pathway in human diseases.

Biological Evaluation in Vitro and in Silico of Azetidin-2-one Derivatives as Potential Anticancer Agents.

Potential anticancer activity of 16 azetidin-2-one derivatives was evaluated showing that compound 6 [N-(p-methoxy-phenyl)-2-(p-methyl-phenyl)-3-phenoxy-azetidin-2-one] presented cytotoxic activity in SiHa cells and B16F10 cells. The caspase-3 assay in B16F10 cells displayed that azetidin-2-one derivatives induce apoptosis. Microarray and molecular analysis showed that compound 6 was involved on specific gene overexpression of cytoskeleton regulation and apoptosis due to the inhibition of some cell cycle genes. From the 16 derivatives, compound 6 showed the highest selectivity to neoplastic cells, it was an inducer of apoptosis, and according to an in silico analysis of chemical interactions with colchicine binding site of human α/ß-tubulin, the mechanism of action could be a molecular interaction involving the amino acids outlining such binding site.

Virtual screening-driven repositioning of etoposide as CD44 antagonist in breast cancer cells.

CD44 is a receptor for hyaluronan (HA) that promotes epithelial-to-mesenchymal transition (EMT), induces cancer stem cell (CSC) expansion, and favors metastasis. Thus, CD44 is a target for the development of antineoplastic agents. In order to repurpose drugs as CD44 antagonists, we performed consensus-docking studies using the HA-binding domain of CD44 and 11,421 molecules. Drugs that performed best in docking were examined in molecular dynamics simulations, identifying etoposide as a potential CD44 antagonist. Ligand competition and cell adhesion assays in MDA-MB-231 cells demonstrated that etoposide decreased cell binding to HA as effectively as a blocking antibody. Etoposide-treated MDA-MB-231 cells developed an epithelial morphology; increased their expression of E-cadherin; and reduced their levels of EMT-associated genes and cell migration. By gene expression analysis, etoposide reverted an EMT signature similarly to CD44 knockdown, whereas other topoisomerase II (TOP2) inhibitors did not. Moreover, etoposide decreased the proportion of CD44+/CD24- cells, lowered chemoresistance, and blocked mammosphere formation. Our data indicate that etoposide blocks CD44 activation, impairing key cellular functions that drive malignancy, thus rendering it a candidate for further translational studies and a potential lead compound in the development of new CD44 antagonists.

An integrated network platform for contextual prioritization of drugs and pathways.

Repurposing of drugs to novel disease indications has promise for faster clinical translation. However, identifying the best drugs for a given pathological context is not trivial. We developed an integrated random walk-based network framework that combines functional biomolecular relationships and known drug-target interactions as a platform for contextual prioritization of drugs, genes and pathways. We show that the use of gene-centric or drug-centric data, such as gene expression data or a phenotypic drug screen, respectively, within this network platform can effectively prioritize drugs and pathways, respectively, to the studied biological context. We demonstrate that various genomic data can be used as contextual cues to effectively prioritize drugs to the studied context, while similarly, phenotypic drug screen data can be used to effectively prioritize genes and pathways to the studied phenotypic context. As a proof-of-principle, we showcase the use of our platform to identify known and novel drug indications against different subsets of breast cancers through contextual prioritization based on genome-wide gene expression, shRNA and drug screen and clinical survival data. The integrated network and associated methods are incorporated into the NetWalker suite for functional genomics analysis ().

Associations of SNPs located at candidate genes to bovine growth traits, prioritized with an interaction networks construction approach.

BACKGROUND: For most domestic animal species, including bovines, it is difficult to identify causative genetic variants involved in economically relevant traits. The candidate gene approach is efficient because it investigates genes that are expected to be associated with the expression of a trait and defines whether the genetic variation present in a population is associated with phenotypic diversity. A potential limitation of this approach is the identification of candidates. This study used a bioinformatics approach to identify candidate genes via a search guided by a functional interaction network. RESULTS: A functional interaction network tool, BosNet, was constructed for Bos taurus. Predictions for candidate genes were performed using the guilt-by-association principle in BosNet. Association analyses identified five novel markers within BosNet-prioritized genes that had significant effects on different growth traits in Charolais and Brahman cattle. CONCLUSIONS: BosNet is an excellent tool for the identification of single nucleotide polymorphisms that are potentially associated with complex traits.

LATS1 and LATS2 phosphorylate CDC26 to modulate assembly of the tetratricopeptide repeat subcomplex of APC/C.

In budding yeast, the Mitotic Exit Network (MEN) regulates anaphase promoting complex/cyclosome (APC/C) via the Dbf2-Cdc14 signaling cascade. Dbf2 kinase phosphorylates and activates Cdc14 phosphatase, which removes the inhibitory phosphorylation of the APC/C cofactor Cdh1. Although each component of the MEN was highly conserved during evolution, there is presently no evidence supporting direct phosphorylation of CDC14 by large tumor suppressor kinase 1 (LATS1), the human counterpart of Dbf2; hence, it is unclear how LATS1 regulates APC/C. Here, we demonstrate that LATS1 phosphorylates the Thr7 (T7) residue of the APC/C component CDC26 directly. Nocodazole-induced phosphorylation of T7 was reduced by knockdown of LATS1 and LATS2 in HeLa cells, indicating that both of these kinases contribute to the phosphorylation of CDC26 in vivo. The T7 residue of CDC26 is critical for its interaction with APC6, a tetratricopeptide repeat-containing subunit of APC/C, and mutation of this residue to Asp (T7D) reduced the interaction of CDC26 with APC6. Replacement of endogenous CDC26 in HeLa cells with exogenous phosphor-mimic T7D-mutated CDC26 increased the elution size of APC/C subunits in a gel filtration assay, implying a change in the APC/C assembly upon phosphorylation of CDC26. Furthermore, T7D-mutated CDC26 promoted the ubiquitination of polo-like kinase 1, a well-known substrate of APC/C. Overall, these results suggest that LATS1/2 are novel kinases involved in APC/C phosphorylation and indicate a direct regulatory link between LATS1/2 and APC/C.

Potential mechanism of action of meso-dihydroguaiaretic acid on Mycobacterium tuberculosis H37Rv.

The isolation and characterization of the lignan meso-dihydroguaiaretic acid (MDGA) from Larrea tridentata and its activity against Mycobacterial tuberculosis has been demonstrated, but no information regarding its mechanism of action has been documented. Therefore, in this study we carry out the gene expression from total RNA obtained from M. tuberculosis H37Rv treated with MDGA using microarray technology, which was validated by quantitative real time polymerase chain reaction. Results showed that the alpha subunit of coenzyme A transferase of M. tuberculosis H37Rv is present in both geraniol and 1-and 2-methylnaphthalene degradation pathways, which are targeted by MDGA. This assumption was supported by molecular docking which showed stable interaction between MDGA with the active site of the enzyme. We propose that inhibition of coenzyme A transferase of M. tuberculosis H37Rv results in the accumulation of geraniol and 1-and 2-methylnaphtalene inside bacteria, causing membrane destabilization and death of the pathogen. The natural product MDGA is thus an attractive template to develop new anti-tuberculosis drugs, because its target is different from those of known anti-tubercular agents.

Anti-Trypanosoma cruzi and anti-leishmanial activity by quinoxaline-7-carboxylate 1,4-di-N-oxide derivatives.

In this work, a novel series of ethyl and methyl quinoxaline-7-carboxylate 1,4-di-N-oxide derivatives were evaluated in vitro on Trypanosoma cruzi trypomastigotes and Leishmania mexicana promastigotes, and cytotoxicity activity in murine macrophages was tested. In silico molecular docking simulations of trypanothione reductase were also done. Three compounds of 33 quinoxaline-7-carboxylate 1,4-di-N-oxide derivatives showed better anti-T. cruzi activity than nifurtimox and beznidazole; two compounds had better anti-leishmanial activity that amphotericin-B, and two compounds showed better activity against both parasites than reference drugs. Compounds M2, M7, M8 and E5, showed low cytotoxic activity on the host cell. The in silico studies suggest that compound M2 is a potential trypanothione reductase inhibitor.

Large scale genome analysis shows that the epitopes for broadly cross-reactive antibodies are predominant in the pandemic 2009 influenza virus A H1N1 strain.

The past pandemic strain H1N1 (A (H1N1)pdm09) has now become a common component of current seasonal influenza viruses. It has changed the pre-existing immunity of the human population to succeeding infections. In the present study, a total of 14,210 distinct sequences downloaded from National Center for Biotechnology Information (NCBI) database were used for the analysis. The epitope compositions in A (H1N1)pdm09, classic seasonal strains, swine strains as well as highly virulent avian strain H5N1, identified with the aid of the Immune Epitope DataBase (IEDB), were compared at genomic level. The result showed that A (H1N1) pdm09 contains the 90% of B-cell epitopes for broadly cross-reactive antibodies (EBCA), which is in consonance with the recent reports on the experimental identification of new epitopes or antibodies for this virus and the binding tests with influenza virus protein HA of different subtypes. Our analysis supports that high proportional EBCA depends on the epitope pattern of A (H1N1)pdm09 virus. This study may be helpful for better understanding of A (H1N1)pdm09 and the production of new influenza vaccines.

A viral-human interactome based on structural motif-domain interactions captures the human infectome.

Protein interactions between a pathogen and its host are fundamental in the establishment of the pathogen and underline the infection mechanism. In the present work, we developed a single predictive model for building a host-viral interactome based on the identification of structural descriptors from motif-domain interactions of protein complexes deposited in the Protein Data Bank (PDB). The structural descriptors were used for searching, in a database of protein sequences of human and five clinically important viruses; therefore, viral and human proteins sharing a descriptor were predicted as interacting proteins. The analysis of the host-viral interactome allowed to identify a set of new interactions that further explain molecular mechanism associated with viral infections and showed that it was able to capture human proteins already associated to viral infections (human infectome) and non-infectious diseases (human diseasome). The analysis of human proteins targeted by viral proteins in the context of a human interactome showed that their neighbors are enriched in proteins reported with differential expression under infection and disease conditions. It is expected that the findings of this work will contribute to the development of systems biology for infectious diseases, and help guide the rational identification and prioritization of novel drug targets.