Erratum: A model system for antiviral siRNA therapeutics using exosome-based delivery.

[This corrects the article DOI: 10.1016/j.omtn.2022.08.011.].

A model system for antiviral siRNA therapeutics using exosome-based delivery.

Emerging viral diseases, such as Ebola, SARS, MERS, and the pathogen causing COVID-19, SARS-CoV-2, present a challenge for the development of therapeutics because of strict biosafety handling procedures and rapid mutation of their genomes. To facilitate the development of an adaptable and testable therapeutic model system, a colostrum exosome-based nanoparticle delivery system, EPM (exosome-PEI matrix), that overcomes stringent biosafety containment, was used to mimic the expression of viral proteins. This system would greatly expand the number of laboratories actively participating in the screening of potential therapeutics. EPM technology can deliver both plasmid DNA and siRNA to both simulate viral gene expression and screen potential antiviral siRNA therapeutics. Using this nanoplatform, three key SARS-CoV-2 proteins (the spike glycoprotein, nucleocapsid, and replicase) were expressed in vitro and in vivo. In vitro, several viral gene-targeting siRNAs were screened to determine knockdown efficiency, with some siRNA duplexes resulting in 80%-95% knockdown of corresponding protein expression. Moreover, in vivo experiments introducing the spike protein and nucleocapsid by EPM resulted in the production of antibodies against the viral antigen, measured up to 45 d after target delivery. Together, these findings support the efficacy of the EPM delivery system to establish a model for screening antiviral therapeutics-reduced biosafety level.

A scalable approach to topographically mediated antimicrobial surfaces based on diamond.

Bio-inspired Topographically Mediated Surfaces (TMSs) based on high aspect ratio nanostructures have recently been attracting significant attention due to their pronounced antimicrobial properties by mechanically disrupting cellular processes. However, scalability of such surfaces is often greatly limited, as most of them rely on micro/nanoscale fabrication techniques. In this report, a cost-effective, scalable, and versatile approach of utilizing diamond nanotechnology for producing TMSs, and using them for limiting the spread of emerging infectious diseases, is introduced. Specifically, diamond-based nanostructured coatings are synthesized in a single-step fabrication process with a densely packed, needle- or spike-like morphology. The antimicrobial proprieties of the diamond nanospike surface are qualitatively and quantitatively analyzed and compared to other surfaces including copper, silicon, and even other diamond surfaces without the nanostructuring. This surface is found to have superior biocidal activity, which is confirmed via scanning electron microscopy images showing definite and widespread destruction of E. coli cells on the diamond nanospike surface. Consistent antimicrobial behavior is also observed on a sample prepared seven years prior to testing date.

Cumin Prevents 17ß-Estradiol-Associated Breast Cancer in ACI Rats.

Breast cancer (BC) is a leading cause of cancer deaths in women in less developed countries and the second leading cause of cancer death in women in the U.S. In this study, we report the inhibition of E2-mediated mammary tumorigenesis by Cuminum cyminum (cumin) administered via the diet as cumin powder, as well as dried ethanolic extract. Groups of female ACI rats were given either an AIN-93M diet or a diet supplemented with cumin powder (5% and 7.5%, w/w) or dried ethanolic cumin extract (1%, w/w), and then challenged with subcutaneous E2 silastic implants (1.2 cm; 9 mg). The first appearance of a palpable mammary tumor was significantly delayed by both the cumin powder and extract. At the end of the study, the tumor incidence was 96% in the control group, whereas only 55% and 45% animals had palpable tumors in the cumin powder and extract groups, respectively. Significant reductions in tumor volume (660 ± 122 vs. 138 ± 49 and 75 ± 46 mm3) and tumor multiplicity (4.21 ± 0.43 vs. 1.16 ± 0.26 and 0.9 ± 0.29 tumors/animal) were also observed by the cumin powder and cumin extract groups, respectively. The cumin powder diet intervention dose- and time-dependently offset E2-related pituitary growth, and reduced the levels of circulating prolactin and the levels of PCNA in the mammary tissues. Mechanistically, the cumin powder diet resulted in a significant reversal of E2-associated modulation in ERα, CYP1A1 and CYP1B1. Further, the cumin powder diet reversed the expression levels of miRNAs (miR-182, miR-375, miR-127 and miR-206) that were highly modulated by E2 treatment. We analyzed the composition of the extract by GC/MS and established cymene and cuminaldehyde as major components, and further detected no signs of gross or systemic toxicity. Thus, cumin bioactives can significantly delay and prevent E2-mediated mammary tumorigenesis in a safe and effective manner, and warrant continued efforts to develop these clinically translatable spice bioactives as chemopreventives and therapeutics against BC.

Exosome-mediated delivery of RNA and DNA for gene therapy.

Gene therapy promises to revolutionize biomedicine and personalized medicine by modulating or compensating the expression of abnormal genes. The biggest obstacle for clinical application is the lack of an effective, non-immunogenic delivery system. We show that bovine colostrum exosomes and polyethyleneimine matrix (EPM) delivers short interfering RNA (siRNA) or plasmid DNA (pDNA) for effective gene therapy. KRAS, a therapeutic focus for many cancers, was targeted by EPM-delivered KRAS siRNA (siKRAS) and inhibited lung tumor growth (>70%) and reduced KRAS expression (50%-80%). Aberrant p53 is another therapeutic focus for many cancers. EPM-mediated introduction of wild-type (WT) p53 pDNA (pcDNA-p53) resulted in p53 expression in p53-null H1299 cells in culture, subcutaneous lung tumor, and tissues of p53-knockout mice. Additionally, chemo-sensitizing effects of paclitaxel were restored by exogenous WT p53 in lung cancer cells. Together, this novel EPM technology represents an effective 'platform' for delivery of therapeutic nucleic acids to treat human disease.

Tender coconut water suppresses hepatic inflammation by activating AKT and JNK signaling pathways in an in vitro model of sepsis.

Tender coconut water (TCW) is a natural plant product rich in phytochemicals and protects against toxic liver injury. However, the mechanism by which TCW inhibits inflammation and tissue damage is unknown. We examined the effect of TCW on primary rat hepatocyte viability, cytokine-induced gene expression and proinflammatory signaling in an in vitro model of sepsis. We observed that TCW improved hepatocyte viability and protected hepatocytes against cytokine-mediated cell death. TCW suppressed IL-1ß-mediated increases in Nos2, Tnf, and Il6 mRNA and increased heme oxygenase 1 (HMOX1) protein. TCW inhibited iNOS expression through activation of AKT and JNK pathways since inhibition of PI3K and JNK signaling reduced TCW's effect on iNOS protein expression and activity. These results demonstrate that TCW reduces proinflammatory gene expression and hepatocyte injury produced by elevated inflammatory cytokines and nitric oxide production.

Transcriptomic response of breast cancer cells to anacardic acid.

Anacardic acid (AnAc), a potential dietary agent for preventing and treating breast cancer, inhibited the proliferation of estrogen receptor α (ERα) positive MCF-7 and MDA-MB-231 triple negative breast cancer cells. To characterize potential regulators of AnAc action, MCF-7 and MDA-MB-231 cells were treated for 6 h with purified AnAc 24:1n5 congener followed by next generation transcriptomic sequencing (RNA-seq) and network analysis. We reported that AnAc-differentially regulated miRNA transcriptomes in each cell line and now identify AnAc-regulated changes in mRNA and lncRNA transcript expression. In MCF-7 cells, 80 AnAc-responsive genes were identified, including lncRNA MIR22HG. More AnAc-responsive genes (886) were identified in MDA-MB-231 cells. Only six genes were commonly altered by AnAc in both cell lines: SCD, INSIG1, and TGM2 were decreased and PDK4, GPR176, and ZBT20 were increased. Modeling of AnAc-induced gene changes suggests that AnAc inhibits monounsaturated fatty acid biosynthesis in both cell lines and increases endoplasmic reticulum stress in MDA-MB-231 cells. Since modeling of downregulated genes implicated NFκB in MCF-7, we confirmed that AnAc inhibited TNFα-induced NFκB reporter activity in MCF-7 cells. These data identify new targets and pathways that may account for AnAc's anti-proliferative and pro-apoptotic activity.

Pas de deux: An Intricate Dance of Anther Smut and Its Host.

The successful interaction between pathogen/parasite and host requires a delicate balance between fitness of the former and survival of the latter. To optimize fitness a parasite/pathogen must effectively create an environment conducive to reproductive success, while simultaneously avoiding or minimizing detrimental host defense response. The association between Microbotryum lychnidis-dioicae and its host Silene latifolia serves as an excellent model to examine such interactions. This fungus is part of a species complex that infects species of the Caryophyllaceae, replacing pollen with the fungal spores. In the current study, transcriptome analyses of the fungus and its host were conducted during discrete stages of bud development so as to identify changes in fungal gene expression that lead to spore development and to identify changes associated with infection in the host plant. In contrast to early biotrophic phase stages of infection for the fungus, the latter stages involve tissue necrosis and in the case of infected female flowers, further changes in the developmental program in which the ovary aborts and a pseudoanther is produced. Transcriptome analysis via Illumina RNA sequencing revealed enrichment of fungal genes encoding small secreted proteins, with hallmarks of effectors and genes found to be relatively unique to the Microbotryum species complex. Host gene expression analyses also identified interesting sets of genes up-regulated, including those involving stress response, host defense response, and several agamous-like MADS-box genes (AGL61 and AGL80), predicted to interact and be involved in male gametophyte development.

Genome-wide miRNA response to anacardic acid in breast cancer cells.

MicroRNAs are biomarkers and potential therapeutic targets for breast cancer. Anacardic acid (AnAc) is a dietary phenolic lipid that inhibits both MCF-7 estrogen receptor α (ERα) positive and MDA-MB-231 triple negative breast cancer (TNBC) cell proliferation with IC50s of 13.5 and 35 µM, respectively. To identify potential mediators of AnAc action in breast cancer, we profiled the genome-wide microRNA transcriptome (microRNAome) in these two cell lines altered by the AnAc 24:1n5 congener. Whole genome expression profiling (RNA-seq) and subsequent network analysis in MetaCore Gene Ontology (GO) algorithm was used to characterize the biological pathways altered by AnAc. In MCF-7 cells, 69 AnAc-responsive miRNAs were identified, e.g., increased let-7a and reduced miR-584. Fewer, i.e., 37 AnAc-responsive miRNAs were identified in MDA-MB-231 cells, e.g., decreased miR-23b and increased miR-1257. Only two miRNAs were increased by AnAc in both cell lines: miR-612 and miR-20b; however, opposite miRNA arm preference was noted: miR-20b-3p and miR-20b-5p were upregulated in MCF-7 and MDA-MB-231, respectively. miR-20b-5p target EFNB2 transcript levels were reduced by AnAc in MDA-MB-231 cells. AnAc reduced miR-378g that targets VIM (vimentin) and VIM mRNA transcript expression was increased in AnAc-treated MCF-7 cells, suggesting a reciprocal relationship. The top three enriched GO terms for AnAc-treated MCF-7 cells were B cell receptor signaling pathway and ribosomal large subunit biogenesis and S-adenosylmethionine metabolic process for AnAc-treated MDA-MB-231 cells. The pathways modulated by these AnAc-regulated miRNAs suggest that key nodal molecules, e.g., Cyclin D1, MYC, c-FOS, PPARγ, and SIN3, are targets of AnAc activity.

Chemoprevention of Rat Mammary Carcinogenesis by Apiaceae Spices.

Scientific evidence suggests that many herbs and spices have medicinal properties that alleviate symptoms or prevent disease. In this study, we examined the chemopreventive effects of the Apiaceae spices, anise, caraway, and celery seeds against 17ß-estrogen (E2)-mediated mammary tumorigenesis in an ACI (August-Copenhagen Irish) rat model. Female ACI rats were given either control diet (AIN 93M) or diet supplemented with 7.5% (w/w) of anise, caraway, or celery seed powder. Two weeks later, one half of the animals in each group received subcutaneous silastic implants of E2. Diet intake and body weight were recorded weekly, and animals were euthanized after 3 and 12 weeks. E2-treatment showed significantly (2.1- and 3.4-fold) enhanced growth of pituitary gland at 3 and 12 weeks, respectively. All test spices significantly offset the pituitary growth by 12 weeks, except celery which was effective as early as three weeks. Immunohistochemical analysis for proliferative cell nuclear antigen (PCNA) in mammary tissues showed significant reduction in E2-mediated mammary cell proliferation. Test spices reduced the circulating levels of both E2 and prolactin at three weeks. This protection was more pronounced at 12 weeks, with celery eliciting the highest effect. RT-PCR and western blot analysis were performed to determine the potential molecular targets of the spices. Anise and caraway diets significantly offset estrogen-mediated overexpression of both cyclin D1 and estrogen receptor α (ERα). The effect of anise was modest. Likewise, expression of CYP1B1 and CYP1A1 was inhibited by all test spices. Based on short-term molecular markers, caraway was selected over other spices based on its enhanced effect on estrogen-associated pathway. Therefore, a tumor-end point study in ACI rats was conducted with dietary caraway. Tumor palpation from 12 weeks onwards revealed tumor latency of 29 days in caraway-treated animals compared with first tumor appearance at 92 days in control group. At the end of the study (25 weeks), the tumor incidence was 96% in the control group compared with only 70% in the caraway group. A significant reduction in tumor volume (661 ± 123 vs. 313 ± 81 mm³) and tumor multiplicity (4.2 ± 0.4 vs. 2.5 ± 0.5 tumors/animal) was also observed in the caraway group compared with the control group. Together, our data show dietary caraway can significantly delay and prevent the hormonal mammary tumorigenesis by modulating different cellular and molecular targets.

Transcriptional analysis of mating and pre-infection stages of the anther smut, Microbotryum lychnidis-dioicae.

Microbotryum lychnidis-dioicae is an obligate biotrophic parasite of the wildflower species Silene latifolia. This dikaryotic fungus, commonly known as an anther smut, requires that haploid, yeast-like sporidia of opposite mating types fuse and differentiate into dikaryotic hyphae that penetrate host tissue as part of the fungal life cycle. Mating occurs under conditions of cool temperatures and limited nutrients. Further development requires host cues or chemical mimics, including a variety of lipids, e.g. phytols. To identify global changes in transcription associated with developmental shifts, RNA-Seq was conducted at several in vitro stages of fungal propagation, i.e. haploid cells grown independently on rich and nutrient-limited media, mated cells on nutrient-limited media as well as a time course of such mated cells exposed to phytol. Comparison of haploid cells grown under rich and nutrient-limited conditions identified classes of genes probably associated with general nutrient availability, including components of the RNAi machinery. Some gene enrichment patterns comparing the nutrient-limited and mated transcriptomes suggested gene expression changes associated with the mating programme (e.g. homeodomain binding proteins, secreted proteins, proteins unique to M. lychnidis-dioicae¸ multicopper oxidases and RhoGEFs). Analysis for phytol treatment compared with mated cells alone allowed identification of genes likely to be involved in the dikaryotic switch (e.g. oligopeptide transporters). Gene categories of particular note in all three conditions included those in the major facilitator superfamily, proteins containing PFAM domains of the secretory lipase family as well as proteins predicted to be secreted, many of which have the hallmarks of fungal effectors with potential roles in pathogenicity.

Anacardic Acid, Salicylic Acid, and Oleic Acid Differentially Alter Cellular Bioenergetic Function in Breast Cancer Cells.

Anacardic acid is a dietary and medicinal phytochemical that inhibits breast cancer cell proliferation and uncouples oxidative phosphorylation (OXPHOS) in isolated rat liver mitochondria. Since mitochondrial-targeted anticancer therapy (mitocans) may be useful in breast cancer, we examined the effect of anacardic acid on cellular bioenergetics and OXPHOS pathway proteins in breast cancer cells modeling progression to endocrine-independence: MCF-7 estrogen receptor α (ERα)+ endocrine-sensitive; LCC9 and LY2 ERα+, endocrine-resistant, and MDA-MB-231 triple negative breast cancer (TNBC) cells. At concentrations similar to cell proliferation IC50 s, anacardic acid reduced ATP-linked oxygen consumption rate (OCR), mitochondrial reserve capacity, and coupling efficiency while increasing proton leak, reflecting mitochondrial toxicity which was greater in MCF-7 compared to endocrine-resistant and TNBC cells. These results suggest tolerance in endocrine-resistant and TNBC cells to mitochondrial stress induced by anacardic acid. Since anacardic acid is an alkylated 2-hydroxybenzoic acid, the effects of salicylic acid (SA, 2-hydroxybenzoic acid moiety) and oleic acid (OA, monounsaturated alkyl moiety) were tested. SA inhibited whereas OA stimulated cell viability. In contrast to stimulation of basal OCR by anacardic acid (uncoupling effect), neither SA nor OA altered basal OCR- except OA inhibited basal and ATP-linked OCR, and increased ECAR, in MDA-MB-231 cells. Changes in OXPHOS proteins correlated with changes in OCR. Overall, neither the 2-hydroxybenzoic acid moiety nor the monounsaturated alky moiety of anacardic acid is solely responsible for the observed mitochondria-targeted anticancer activity in breast cancer cells and hence both moieties are required in the same molecule for the observed effects. J. Cell. Biochem. 117: 2521-2532, 2016. © 2016 Wiley Periodicals, Inc.

Potent Chemopreventive/Antioxidant Activity Detected in Common Spices of the Apiaceae Family.

Spices are used worldwide, particularly in the Asian and Middle Eastern countries, and considered protective against degenerative diseases, including cancer. Here, we report the efficacy of aqueous and non-aqueous extracts of 11 Apiaceae spices for free radical-scavenging activity and to inhibit cytochrome P450s in two separate reactions involving: 1) 4-hydroxy-17ß-estradiol (4E2), DNA, and CuCl2 and 2) 17ß-estradiol, rat liver microsomes, cofactors, DNA and CuCl2. Oxidative DNA adducts resulting from redox cycling of 4E2 were analyzed by (32)P-postlabeling. Aqueous (5 mg/ml) and non-aqueous extracts (6 mg/ml) substantially inhibited (83-98%) formation of DNA adducts in the microsomal reaction. However, in nonmicrosomal reaction, only aqueous extracts showed the inhibitory activity (83-96%). Adduct inhibition was also observed at five-fold lower concentrations of aqueous extracts of cumin (60%) and caraway (90%), and 10-fold lower concentrations of carrot seeds (76%) and ajowan (90%). These results suggests the presence of 2 groups of phytochemicals: polar compounds that have free radical-scavenging activity and lipophilic compounds that selectively inhibit P450 activity associated with estrogen metabolism. Because most of these Apiaceae spices are used widely with no known toxicity, the phytochemicals from the Apiaceae spices used in foods may be potentially protective against estrogen-mediated breast cancer.

Sex and parasites: genomic and transcriptomic analysis of Microbotryum lychnidis-dioicae, the biotrophic and plant-castrating anther smut fungus.

BACKGROUND: The genus Microbotryum includes plant pathogenic fungi afflicting a wide variety of hosts with anther smut disease. Microbotryum lychnidis-dioicae infects Silene latifolia and replaces host pollen with fungal spores, exhibiting biotrophy and necrosis associated with altering plant development. RESULTS: We determined the haploid genome sequence for M. lychnidis-dioicae and analyzed whole transcriptome data from plant infections and other stages of the fungal lifecycle, revealing the inventory and expression level of genes that facilitate pathogenic growth. Compared to related fungi, an expanded number of major facilitator superfamily transporters and secretory lipases were detected; lipase gene expression was found to be altered by exposure to lipid compounds, which signaled a switch to dikaryotic, pathogenic growth. In addition, while enzymes to digest cellulose, xylan, xyloglucan, and highly substituted forms of pectin were absent, along with depletion of peroxidases and superoxide dismutases that protect the fungus from oxidative stress, the repertoire of glycosyltransferases and of enzymes that could manipulate host development has expanded. A total of 14% of the genome was categorized as repetitive sequences. Transposable elements have accumulated in mating-type chromosomal regions and were also associated across the genome with gene clusters of small secreted proteins, which may mediate host interactions. CONCLUSIONS: The unique absence of enzyme classes for plant cell wall degradation and maintenance of enzymes that break down components of pollen tubes and flowers provides a striking example of biotrophic host adaptation.

Ustilago maydis phosphodiesterases play a role in the dimorphic switch and in pathogenicity.

Components of the cAMP (cyclic AMP) signalling cascades are conserved from fungi to humans, and are particularly important for fungal dimorphism and pathogenicity. Previous work has described two phosphodiesterases, UmPde1 and UmPde2, in Ustilago maydis which show strong phosphodiesterase activity. We further characterized the biological function(s) of these phosphodiesterases in U. maydis. Specifically, we examined their possible role(s) in regulation of the cAMP-dependent protein kinase A (PKA) pathway and their roles in filamentous growth and pathogenicity. We found that UmPde1, which shares 35 % similarity with Cryptococcus neoformans Pde1, also displays functional homology with this enzyme. UmPde1 complements the capsule-formation defect of C. neoformans strains deleted for Pde1. In U. maydis, the cell morphology of the umpde1 deletion mutant resembled the multiple budding phenotypes seen with the ubc1 mutant, which lacks the regulatory subunit of PKA. Interestingly, on low-ammonium medium, umpde2 deletion strains showed a reduction in filamentation that was comparable to that of ubc1 deletion strains; however, umpde1 deletion strains showed normal filamentation on low-ammonium medium. Furthermore, both the ubc1 deletion strain in which the PKA pathway was constitutively active and the umpde1 deletion strains were significantly reduced in pathogenicity, while the umpde2 deletion strains showed a trend for reduced pathogenicity compared with wild-type strains. These data support a role for the phosphodiesterases UmPde1 and UmPde2 in regulating the U. maydis cAMP-dependent PKA pathway through modulation of cAMP levels, thus affecting dimorphic growth and pathogenicity.

Anti-proliferative activity and protection against oxidative DNA damage by punicalagin isolated from pomegranate husk.

Ellagitannins are the most abundant polyphenols in pomegranate (Punica granatum) husk and contribute greatly towards its biological properties. A pre-enriched pomegranate husk powder was extracted with water and then further purified by an Amberlite XAD-16 column. Punicalagin (PC) anomers were eluted using a gradient of methanol and water. Fractions eluted with 20% and 25% methanol yielded 1.08 g of light brown powder (purity > 97%) from a total of 40 g of extract. This fraction was identified as PC by HPLC-UV using reference compounds and confirmed by FTICR-MS analysis. PC (10-40 µM) was found to significantly inhibit oxidative DNA products, about 70% inhibition at 40 µM (p=0.0017), resulting from Cu2+-catalyzed redox cycling of 4-hydroxy-17ß-estradiol as analyzed by 32P-postlabeling. Evidence of high antioxidant activity of PC was also obtained based on ORAC assay (1556±79 µmol of TE/g), as well as by 2,2'-azino-bis (3-ethylbenzthiazoline-6-sulphonic acid) (ABTS)-, 2,2-diphenyl-1-picrylhydrazyl (DPPH)-, hydrogen peroxide (H2O2) scavenging and ferrous ion-chelating activities (IC50=1.1, 17.1, 24 and 45.4 µg/ml, respectively). Further, PC exhibited strong anti-proliferative activity against the human lung, breast and cervical cancer cell lines. Together, these data suggest that PC can be isolated in its purified form by simple column chromatography, inhibits oxidative DNA damage and possesses high anti-proliferative activity.

Pharmacokinetics of moxidectin in the southern hairy-nosed wombat (Lasiorhinus latifrons).

Sarcoptic mange, caused by Sarcoptes scabiei var. wombati, could be a significant threat to populations of southern hairy-nosed wombats (Lasiorhinus latifrons; SHNW) in Australia. Treatment is currently based on the off-label use of various parasiticidal drugs, with limited clinical efficacy trials. Our primary aim was to determine the pharmacokinetic parameters of a macrocyclic lactone, moxidectin, to assist in the development of effective treatment protocols. Pharmacokinetic parameters were determined in four female SHNW following a single subcutaneous injection of 0.2 mg/kg moxidectin. Blood samples were collected for 38 days following injection (August-September 2008), for analysis using liquid chromatography and tandem mass spectrometry. The mean peak plasma concentration occurred at 13.6 hr, with a mean peak plasma level of 98.6 ng/ml. The mean elimination half-life was 5.03 days, resulting in a mean area under the curve of 377 ng.day/ml. The peak plasma moxidectin concentration was higher than that seen in livestock species but the plasma elimination half-life was shorter. This study suggests that a single injection of 0.2 mg/kg moxidectin may not be sufficient to clear a mange infection in this species.

Anacardic acid inhibits estrogen receptor alpha-DNA binding and reduces target gene transcription and breast cancer cell proliferation.

Anacardic acid (AnAc; 2-hydroxy-6-alkylbenzoic acid) is a dietary and medicinal phytochemical with established anticancer activity in cell and animal models. The mechanisms by which AnAc inhibits cancer cell proliferation remain undefined. AnAc 24:1(omega5) was purified from geranium (Pelargonium x hortorum) and shown to inhibit the proliferation of estrogen receptor alpha (ERalpha)-positive MCF-7 and endocrine-resistant LCC9 and LY2 breast cancer cells with greater efficacy than ERalpha-negative primary human breast epithelial cells, MCF-10A normal breast epithelial cells, and MDA-MB-231 basal-like breast cancer cells. AnAc 24:1(omega5) inhibited cell cycle progression and induced apoptosis in a cell-specific manner. AnAc 24:1(omega5) inhibited estradiol (E(2))-induced estrogen response element (ERE) reporter activity and transcription of the endogenous E(2) target genes pS2, cyclin D1, and cathepsin D in MCF-7 cells. AnAc 24:1(omega5) did not compete with E(2) for ERalpha or ERbeta binding, nor did AnAc 24:1(omega5) reduce ERalpha or ERbeta steady-state protein levels in MCF-7 cells; rather, AnAc 24:1(omega5) inhibited ER-ERE binding in vitro. Virtual screening with the molecular docking software Surflex evaluated AnAc 24:1(omega5) interaction with ERalpha ligand binding (LBD) and DNA binding (DBD) domains in conjunction with experimental validation. Molecular modeling revealed AnAc 24:1(omega5) interaction with the ERalpha DBD but not the LBD. Chromatin immunoprecipitation experiments revealed that AnAc 24:1(omega5) inhibited E(2)-ERalpha interaction with the endogenous pS2 gene promoter region containing an ERE. These data indicate that AnAc 24:1(omega5) inhibits cell proliferation, cell cycle progression, and apoptosis in an ER-dependent manner by reducing ER-DNA interaction and inhibiting ER-mediated transcriptional responses.

Concurrent macular oedema and optic neuritis in Crohn's disease with varied response to steroids.

A patient with a 10-year history of Crohn's disease presented to our ophthalmology service with symptoms of acute anterior uveitis. Previous episodes had been treated successfully with topical steroids.However, on this occasion after treatment with topical steroids, the patient developed macular oedema with concurrent optic neuritis in the affected eye.Furthermore, optic neuritis persisted despite oral steroid treatment contrary to expectations based on current literature. After 6 months of treatment, optic disc oedema had markedly improved but was replaced by sectoral disc pallor.

Two phosphodiesterases from ustilago maydis share structural and biochemical properties with non-fungal phosphodiesterases.

The dependence of Protein Kinase A (PKA) activity on cAMP levels is an important facet of the dimorphic switch between budding and filamentous growth as well as for pathogenicity in some fungi. To better understand these processes in the pathogenic fungus Ustilago maydis, we characterized the structure and biochemical functions of two phosphodiesterase (PDE) genes. Phosphodiesterases are enzymes involved in cAMP turnover and thus, contribute to the regulation of the cAMP-PKA signaling pathway. Two predicted homologs of PDEs were identified in the genome of U. maydis and hypothesized to be involved in cAMP turnover, thus regulating activity of the PKA catalytic subunit. Both umpde1 and umpde2 genes contain domains associated with phosphodiesterase activity predicted by InterPro analysis. Biochemical characterization of recombinantly produced UmPde1 (U. maydis Phosphodiesterase I) and UmPde2 demonstrated that both enzymes have phosphodiesterase activity in vitro, yet neither was inhibited by the phosphodiesterase inhibitor IBMX. Moreover, UmPde1 is specific for cAMP, while UmPde2 has broader substrate specificity, utilizing cAMP and cGMP as substrates. In addition, UmPde2 was also found to have nucleotide phosphatase activity that was higher with GMP compared to AMP. These results demonstrate that UmPde1 is a bona fide phosphodiesterase, while UmPde2 has more general activity as a cyclic nucleotide phosphodiesterase and/or GMP/AMP phosphatase. Thus, UmPde1 and UmPde2 likely have important roles in cell morphology and development and share some characteristics with a variety of non-fungal phosphodiesterases.