Rewiring of master transcription factor cistromes during high-grade serous ovarian cancer development.

The transcription factors MECOM, PAX8, SOX17 and WT1 are candidate master regulators of high-grade serous 'ovarian' cancer (HGSC), yet their cooperative role in the hypothesized tissue of origin, the fallopian tube secretory epithelium (FTSEC) is unknown. We generated 26 epigenome (CUT&TAG, CUT&RUN, ATAC-seq and HiC) data sets and 24 profiles of RNA-seq transcription factor knock-down followed by RNA sequencing in FTSEC and HGSC models to define binding sites and gene sets regulated by these factors in cis and trans. This revealed that MECOM, PAX8, SOX17 and WT1 are lineage-enriched, super-enhancer associated master regulators whose cooperative DNA-binding patterns and target genes are re-wired during tumor development. All four TFs were indispensable for HGSC clonogenicity and survival but only depletion of PAX8 and WT1 impaired FTSEC cell survival. These four TFs were pharmacologically inhibited by transcriptional inhibitors only in HGSCs but not in FTSECs. Collectively, our data highlights that tumor-specific epigenetic remodeling is tightly related to MECOM, PAX8, SOX17 and WT1 activity and these transcription factors are targetable in a tumor-specific manner through transcriptional inhibitors.

The "Ins and Outs" of Prostate Specific Membrane Antigen (PSMA) as Specific Target in Prostate Cancer Therapy.

Prostate-specific membrane antigen (PSMA) is expressed in epithelial cells of the prostate gland and is strongly upregulated in prostatic adenocarcinoma, with elevated expression correlating with metastasis, progression, and androgen independence. Because of its specificity, PSMA is a major target of prostate cancer therapy; however, detectable levels of PSMA are also found in other tissues, especially in salivary glands and kidney, generating bystander damage of these tissues. Antibody target therapy has been used with relative success in reducing tumor growth and prostate specific antigen (PSA) levels. However, since antibodies are highly stable in plasma, they have prolonged time in circulation and accumulate in organs with an affinity for antibodies such as bone marrow. For that reason, a second generation of PSMA targeted therapeutic agents has been developed. Small molecules and minibodies have had promising clinical trial results, but concerns about their specificity had arisen with side effects due to accumulation in salivary glands and kidneys. Herein we study the specificity of small molecules and minibodies that are currently being clinically tested. We observed a high affinity of these molecules for PSMA in prostate, kidney and salivary gland, suggesting that their effect is not prostate specific. The search for specific prostate target agents must continue so as to optimally treat patients with prostate cancer, while minimizing deleterious effects in other PSMA expressing tissues.

Telomere Architecture Correlates with Aggressiveness in Multiple Myeloma.

The prognosis of multiple myeloma (MM), an incurable B-cell malignancy, has significantly improved through the introduction of novel therapeutic modalities. Myeloma prognosis is essentially determined by cytogenetics, both at diagnosis and at disease progression. However, for a large cohort of patients, cytogenetic analysis is not always available. In addition, myeloma patients with favorable cytogenetics can display an aggressive clinical course. Therefore, it is necessary to develop additional prognostic and predictive markers for this disease to allow for patient risk stratification and personalized clinical decision-making. Genomic instability is a prominent characteristic in MM, and we have previously shown that the three-dimensional (3D) nuclear organization of telomeres is a marker of both genomic instability and genetic heterogeneity in myeloma. In this study, we compared in a longitudinal prospective study blindly the 3D telomeric profiles from bone marrow samples of 214 initially treatment-naïve patients with either monoclonal gammopathy of undetermined significance (MGUS), smoldering multiple myeloma (SMM), or MM, with a minimum follow-up of 5 years. Here, we report distinctive 3D telomeric profiles correlating with disease aggressiveness and patient response to treatment in MM patients, and also distinctive 3D telomeric profiles for disease progression in smoldering multiple myeloma patients. In particular, lower average intensity (telomere length, below 13,500 arbitrary units) and increased number of telomere aggregates are associated with shorter survival and could be used as a prognostic factor to identify high-risk SMM and MM patients.

p53 CRISPR Deletion Affects DNA Structure and Nuclear Architecture.

The TP53 gene is a key tumor suppressor. Although the tumor suppressor p53 was one of the first to be characterized as a transcription factor, with its main function potentiated by its interaction with DNA, there are still many unresolved questions about its mechanism of action. Here, we demonstrate a novel role for p53 in the maintenance of nuclear architecture of cells. Using three-dimensional (3D) imaging and spectral karyotyping, as well as super resolution microscopy of DNA structure, we observe significant differences in 3D telomere signatures, DNA structure and DNA-poor spaces as well gains or losses of chromosomes, between normal and tumor cells with CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)-deleted or wild-type TP53. Additionally, treatment with Nutlin-3 results in differences in nuclear architecture of telomeres in wild-type but not in p53 knockout MCF-7 (Michigan Cancer Foundation-7) cells. Nutlin-3 binds to the p53-binding pocket of mouse double minute 2 (MDM2) and blocks the p53-MDM2 interaction. Moreover, we demonstrate that another p53 stabilizing small molecule, RITA (reactivation of p53 and induction of tumor cell apoptosis), also induces changes in 3D DNA structure, apparently in a p53 independent manner. These results implicate p53 activity in regulating nuclear organization and, additionally, highlight the divergent effects of the p53 targeting compounds Nutlin-3 and RITA.

Distinct Nuclear Organization of Telomeresand Centromeres in Monoclonal Gammopathyof Undetermined Significance and Multiple Myeloma.

Both multiple myeloma (MM) and its precursor state of monoclonal gammopathy of undetermined significance (MGUS) are characterized by an infiltration of plasma cells into the bone marrow, but the mechanisms underlying the disease progression remain poorly understood. Previous research has indicated that 3D nuclear telomeric and centromeric organization may represent important structural indicators for numerous malignancies. Here we corroborate with previously noted differences in the 3D telomeric architecture and report that modifications in the nuclear distribution of centromeres may serve as a novel structural marker with potential to distinguish MM from MGUS. Our findings improve the current characterization of the two disease stages, providing two structural indicators that may become altered in the progression of MGUS to MM.

Transcriptomic study on persistence and survival of Listeria monocytogenes following lethal treatment with nisin.

OBJECTIVES: The aim of this study was to determine gene expression associated with the persistence of a Listeria monocytogenes stationary-phase population when facing lethal nisin treatment. METHODS: RNA-Seq analysis was used for gene expression profiling of persister cells in nutrient-rich medium (persister TN) compared with untreated cells (non-persister). The results were confirmed using reverse transcription quantitative PCR (RT-qPCR). RESULTS: Functional genes associated with the persister population were identified in multiple systems, such as heat-shock-related stress response, cell wall synthesis, ATP-binding cassette (ABC) transport system, phosphotransferase system (PTS) and SOS/DNA repair. CONCLUSIONS: This study pointed to genetic regulation of persister cells exposed to lethal nisin concentrations and provides some insight into possible mechanisms of impeding bacterial persistence.

Smart Sensing System for the Prognostic Monitoring of Bone Health.

The objective of this paper is to report a novel non-invasive, real-time, and label-free smart assay technique for the prognostic detection of bone loss by electrochemical impedance spectroscopy (EIS). The proposed system incorporated an antibody-antigen-based sensor functionalization to induce selectivity for the C-terminal telopeptide type one collagen (CTx-I) molecules-a bone loss biomarker. Streptavidin agarose was immobilized on the sensing area of a silicon substrate-based planar sensor, patterned with gold interdigital electrodes, to capture the antibody-antigen complex. Calibration experiments were conducted with various known CTx-I concentrations in a buffer solution to obtain a reference curve that was used to quantify the concentration of an analyte in the unknown serum samples. Multivariate chemometric analyses were done to determine the performance viability of the developed system. The analyses suggested that a frequency of 710 Hz is the most discriminating regarding the system sensitivity. A detection limit of 0.147 ng/mL was achieved for the proposed sensor and the corresponding reference curve was linear in the range of 0.147 ng/mL to 2.669 ng/mL. Two sheep blood samples were tested by the developed technique and the results were validated using enzyme-linked immunosorbent assay (ELISA). The results from the proposed technique match those from the ELISA.

Rapid and molecular selective electrochemical sensing of phthalates in aqueous solution.

Reported research work presents real time non-invasive detection of phthalates in spiked aqueous samples by employing electrochemical impedance spectroscopy (EIS) technique incorporating a novel interdigital capacitive sensor with multiple sensing thin film gold micro-electrodes fabricated on native silicon dioxide layer grown on semiconducting single crystal silicon wafer. The sensing surface was functionalized by a self-assembled monolayer of 3-aminopropyltrietoxysilane (APTES) with embedded molecular imprinted polymer (MIP) to introduce selectivity for the di(2-ethylhexyl) phthalate (DEHP) molecule. Various concentrations (1-100 ppm) of DEHP in deionized MilliQ water were tested using the functionalized sensing surface to capture the analyte. Frequency response analyzer (FRA) algorithm was used to obtain impedance spectra so as to determine sample conductance and capacitance for evaluation of phthalate concentration in the sample solution. Spectrum analysis algorithm interpreted the experimentally obtained impedance spectra by applying complex nonlinear least square (CNLS) curve fitting in order to obtain electrochemical equivalent circuit and corresponding circuit parameters describing the kinetics of the electrochemical cell. Principal component analysis was applied to deduce the effects of surface immobilized molecular imprinted polymer layer on the evaluated circuit parameters and its electrical response. The results obtained by the testing system were validated using commercially available high performance liquid chromatography diode array detector system.

Enterocins in food preservation.

The Enterococcus genus, a member of the Lactic Acid Bacteria (LAB) is found in various environments, but more particularly in the intestines of humans and other animals. Although sometimes associated with pathogenicity these bacteria have many benefits. They have been found in traditional artisanal fermented products, are used as probiotic cultures and nowadays extensively studied for the production of bacteriocins--the enterocins. Many of these enterocins have been found to be active against Listeria monocytogenes, and a few have also been reported to be active even against Gram negative bacteria, an unusual property for the bacteriocins produced by LAB. These properties have resulted in many studies describing the use of enterocins as preservatives in foods of animal and vegetable origin. This review covers the most recent information on the use of enterocins as food preservatives, either produced in-situ by the addition of enterocin producing strains or as external preservatives in the form of purified or semi-purified extracts, to prevent the growth of spoilage and pathogenic microorganisms.

Antimicrobial and immunomodulatory activities of an ovine proline/arginine-rich cathelicidin.

In in vitro co-culture experiments, the ovine-derived cathelicidin OaBac5mini showed antimicrobial activity against Escherichia coli cells and modulated production of a cytokine by a mammalian inflammatory cell type (macrophage). Using atomic force microscopy, the morphology of peptide-treated E. coli bacteria showed no cell lysis, indicating an intracellular mode of action of the peptide leading to bacterial cell inhibition. At a concentration of 50microg/mL OaBac5mini, the peptide suppressed production of the inflammatory cytokine interleukin-12 by murine J774A cells that had been stimulated with Staphylococcus aureus strain Cowan; levels of other cytokines were unaffected. Thus, certain cationic peptides can enter and disrupt invading Gram-negative pathogens and may be able to modulate inflammatory responses induced by Gram-positive bacterial products.

Synergistic effects of ovine-derived cathelicidins and other antimicrobials against Escherichia coli O157:H7 and Staphylococcus aureus 1056 MRSA.

Synergistic effects of ovine-derived cathelicidins SMAP29 and OaBac5mini with the antimicrobials polymyxin B, lysozyme, nisin and lactoferrin were investigated against E. coli O157:H7 and S. aureus 1056 MRSA. Lysozyme showed synergy against E. coli O157:H7 with SMAP29, polymyxin B and lactoferrin. Synergy was also found between SMAP29 and lactoferrin against this host. Against S. aureus 1056 MRSA, lysozyme showed synergy with OaBac5mini, polymyxin B and nisin, while synergy was also found between nisin and OaBac5mini and polymyxin B. Other combinations of the antimicrobials were either additive or non-synergistic.

Antimicrobial fragments of the pro-region of cathelicidins and other immune peptides.

In addition to the numerous cathelicidin peptides that are associated with the antimicrobial activity exhibited by a crude extract from ovine blood, a further three peptides with antimicrobial activity have been identified. These were part of the precursor cathelin domain of cathelicidins, a large fragment of platelet factor 4 and a small peptide similar to signal peptide of the T-cell glycoprotein CD4 precursor. Fragments of proteins that are involved in protecting the host from infection may have a secondary purpose as antimicrobial agents once they have carried out their primary purpose and are cleaved the main protein.

Effects of cations on antimicrobial activity of ostricacins-1 and 2 on E. coli O157:H7 and S. aureus 1056MRSA.

Ostricacin-1 and ostricacin-2 (Osp-1 and Osp-2) were beta-defensins antimicrobial peptides that were purified from ostrich leukocytes using a cation-exchange column and a semi-prep RP-HPLC column. Both ostricacins were subjected to increased concentrations of monovalent cations (K(+) and Na(+)) and divalent cations (Ca(2+) and Mg(2+)) in order to investigate the effect of cations on the activity of these ostricacins on Gram-negative bacteria and Gram-positive bacteria. The radial diffusion assay method showed that both ostricacins were sensitive to the presence of cations. The divalent cations showed more antagonized effect on the activity against Gram-negative bacteria than the monovalent cations, as the ostricacins lost ability to inhibit bacterial growth at very low concentration (5 mM). When viewed in the context of other defensins activity, our data support a hypothesis that defensins' overall net positive charge determine the sensitivity to cations.

Mechanisms of action of ostrich beta-defensins against Escherichia coli.

To understand their mechanism of antimicrobial activity against Gram-negative bacteria, ostrich beta-defensins, ostricacins-1 and 2 (Osp-1 and Osp-2), were compared with those of sheep myeloid antimicrobial peptide (SMAP)-29 and human neutrophil peptide (HNP)-1, well-characterized sheep alpha-helical and human alpha-defensin peptides, respectively. Fluorescence-based biochemical assays demonstrated that the ostricacins bound lipopolysaccharides and disrupted both outer and cytoplasmic membrane integrity. The ostricacins' permeabilizing ability was weaker than that of SMAP-29, but stronger than HNP-1. As ostricacins have previously shown the ability to inhibit bacterial growth, these peptides were suggested to be bacteriostatic to Gram-negative bacteria, which are caused by the interaction between the peptides and cytoplasmic targets causing the inhibition of DNA, RNA, and protein synthesis as well as enzymatic activities. These findings indicated promising possibilities for the peptides to be used in the development of therapeutic and topical products.

Identification of three novel ostricacins: an update on the phylogenetic perspective of beta-defensins.

Three new beta-defensins, ostricacins-2, 3 and 4 (Osp-2, 3 and 4), have been successfully purified and characterised from ostrich heterophils in addition to ostricacin-1 (Osp-1). These peptides are composed of 36-42 amino acids with a molecular weight range of 4.70-4.98 kDa. In vitro, Osp-1, 3 and 4 were active against Escherichia coli O157:H7 and Staphylococcus aureus 1056 MRSA, whilst Osp-2 was active against bacterial strains plus the yeast Candida albicans 3153A. Minimal inhibitory concentrations of the three ostricacins ranged from 0.96 microg/mL to 12.03 microg/mL. Comparison with the known beta-defensins from mammalian and other avian species revealed that the four ostricacins shared eight conserved residues (six cysteines and two glycines), identified as the 'beta-defensin core motif'. Comparisons of the sequence also indicated that beta-defensins could have originated from a common beta-defensin-like ancestor that occurred before avian and mammalian lines diverged.

Isolation and characterisation of antimicrobial peptides from deer neutrophils.

The New Zealand deer industry is the largest and most advanced in the world. Antimicrobial peptides have been isolated from a wide range of organisms, but as yet there have been no reports on any from deer. This work investigates the antimicrobial activity and characterisation of peptides isolated from Cervus elaphus blood. It was found that deer blood contains proline/arginine-rich cathelicidins, similar to Bac5 peptides isolated from cattle, sheep and goats. A beta-defensin was also isolated that had a conserved amino acid sequence and mass similar to bovine neutrophil beta-defensins. The cathelicidin displayed strong activity against Gram-negative bacteria, but lesser activity against Gram-positive bacteria and yeast, whilst the beta-defensin showed good activity against all three test organisms.

The role of isocitrate lyase and the glyoxylate cycle in Escherichia coli growing under glucose limitation.

Escherichia coli changes its metabolism in response to environmental circumstances, and metabolic adaptations are evident in hungry bacteria growing slowly in glucose-limited chemostats. The role of isocitrate lyase (AceA) was examined in E. coli growing under glucose limitation. AceA activity was elevated in a strain-dependent manner in the commonly used E. coli K-12 laboratory strains MG1655 and MC4100, but an aceA disruption surprisingly increased fitness under glucose limitation in both strains. However, in bacteria adapted to limiting glucose in long-term chemostats, mutations outside aceA changed its role from a negative to a positive influence. These results suggest that a recently proposed pathway of central metabolism involving the glyoxylate cycle enzymes is redundant in wild-type bacteria, but may take on a beneficial role after context adaptation. Interestingly, the aceA gene sequence did not alter during prolonged selection, so mutations in unidentified genes changed the metabolic context of unaltered AceA from a negative to a positive influence in bacteria highly adapted to limiting glucose.

Factors affecting the antimicrobial activity of ovine-derived cathelicidins against E. coli 0157:H7.

Antimicrobial peptides extracted from ovine neutrophils have potential to be high-value by-products of the lamb industry as, for example, a biopreservative for chilled lamb products. This work was carried out to determine the conditions in which ovine peptides are most effective and to assist in product development. The activities of three synthetic ovine-derived antimicrobial peptides tested were not significantly affected by pH or temperature. However, they exhibited decreased activity at high ionic strengths and in the presence of divalent cations. The three peptides worked better in combination than individually.

Investigation of morphological changes to Staphylococcus aureus induced by ovine-derived antimicrobial peptides using TEM and AFM.

The effect of two ovine-derived peptides on the morphology of Staphylococcus aureus NCTC 4163 cells were investigated using transmission electron microscopy and atomic force microscopy. Both techniques showed that SMAP29, an alpha-helical peptide, induced cell lysis, whereas OaBac5mini, a proline/arginine-rich peptide, caused no observable morphological changes. This is consistent with previous experimental work which indicated that SMAP29 caused cell death and induced cell lysis, whereas OaBac5mini acted by interacting with the inner cellular contents.