CIRCADIAN CLOCK ASSOCIATED1 (CCA1) and the Circadian Control of Stomatal Aperture.

The endogenous circadian (∼24 h) system allows plants to anticipate and adapt to daily environmental changes. Stomatal aperture is one of the many processes under circadian control; stomatal opening and closing occurs under constant conditions, even in the absence of environmental cues. To understand the significance of circadian-mediated anticipation in stomatal opening, we have generated SGC (specifically guard cell) Arabidopsis (Arabidopsis thaliana) plants in which the oscillator gene CIRCADIAN CLOCK ASSOCIATED1 (CCA1) was overexpressed under the control of the guard-cell-specific promoter, GC1. The SGC plants showed a loss of ability to open stomata in anticipation of daily dark-to-light changes and of circadian-mediated stomatal opening in constant light. We observed that under fully watered and mild drought conditions, SGC plants outperform wild type with larger leaf area and biomass. To investigate the molecular basis for circadian control of guard cell aperture, we used large-scale qRT-PCR to compare circadian oscillator gene expression in guard cells compared with the "average" whole-leaf oscillator and examined gene expression and stomatal aperture in several lines of plants with misexpressed CCA1 Our results show that the guard cell oscillator is different from the average plant oscillator. Moreover, the differences in guard cell oscillator function may be important for the correct regulation of photoperiod pathway genes that have previously been reported to control stomatal aperture. We conclude by showing that CONSTANS and FLOWERING LOCUS T, components of the photoperiod pathway that regulate flowering time, also control stomatal aperture in a daylength-dependent manner.

Plasma Activation of a Breast Implant Shell in Conjunction With Antibacterial Irrigants Enhances Antibacterial Activity.

BACKGROUND: Infection and capsular contracture are two of the most significant complications of breast-implant surgery. Both complications are associated with bacterial contamination of the implant surface. Plasma activation of the surface of a silicone breast implant changes its surface properties from water repelling (hydrophobic) to water absorbing (hydrophilic), thus making it possible for antibacterial irrigants to temporarily adsorb onto the implant surface. OBJECTIVES: To support our hypothesis that by changing the surface properties we could render antibacterial irrigation more effective in inhibiting bacterial growth on a breast implant shell. METHODS: An in vitro study using silicone discs cut from a textured silicone breast implant shell was performed by treating some of the discs with plasma activation and then exposing the discs to contamination with either Staphylococcus aureus or Pseudomonas aeruginosa and then variously treating the discs with 10% povidone iodine, Cefazolin, or Gentamicin. Bacterial contamination was verified and counted using contact plates as well as culture media. RESULTS: Plasma activation changed the wetting properties of the disc's surface from hydrophobic to hydrophilic. Nonplasma activated contaminated discs demonstrated clear bacterial growth both in the untreated group and in the antibacterial-treated group. Combining antibacterial treatment with plasma activation resulted in complete inhibition of bacterial growth in each of the groups treated with antibacterial irrigants. CONCLUSIONS: Combining plasma activation with topical antibacterial irrigants can inhibit the growth of bacteria on implant shell discs. By changing the properties of the surface from hydrophobic to hydrophilic, the adsorption of the antibacterial irrigants is enhanced.

Gonadotrophin-releasing hormone agonists for fertility preservation: unraveling the enigma?

STUDY QUESTION: Can gonadotrophin-releasing hormone agonists (GnRH-a) preserve long-term fertility when administered prior to and concomitantly with chemotherapy? SUMMARY ANSWER: GnRH-a display a differential protective effect on fertility, depending upon the specific chemotherapy-induced mechanism of ovarian injury. WHAT IS KNOWN ALREADY: The role of GnRH-a in fertility preservation has been constantly debated and their use is considered experimental due to conflicting clinical evidence and paucity of data regarding their mechanism for ovarian protection. STUDY DESIGN, SIZE, DURATION: In vivo model: 7-8 weeks old imprinting control region (ICR) mice were injected with GnRH-a (Leuprolide-acetate) or saline prior to and concomitantly with cyclophosphamide, doxorubicin or saline and sacrificed at various time-points on a longitudinal follow-up; 24 h (n = 36), 1 week (n = 40), 1 month (n = 36) and 9 months (n = 66) post chemotherapy treatment. Blood samples were drawn on Day 0 and on a monthly basis after chemotherapy treatment. On the day of sacrifice, blood samples were drawn and ovaries excised and processed for either immunohistochemistry (IHC), protein or RNA extraction. In vitro model: 21-23 days old Wistar-derived rats were sacrificed, their ovaries excised and primary granulosa cells (PGC) were either isolated for in vitro culture, or processed for immunofluorescence (IF) as well as for protein or RNA extraction. MATERIALS, SETTING, METHODS: Ovarian reserve was estimated by serial measurements of serum anti-mullerian hormone (AMH), quantified by the AMH Gen II ELISA assay. Ovarian AMH and phosphorylated Akt (pAkt) were detected by immunoblotting. Vascular endothelial growth factor (VEGF) was measured by quantitative PCR. Ovarian GnRH receptor (GnRHR), AMH and CD34 were visualized by IHC, and apoptosis was evaluated using TdT (terminal deoxynucleotidyl transferase)-mediated dUDP nick-end labeling (TUNEL). MAIN RESULTS AND THE ROLE OF CHANCE: Cyclophosphamide-induced ovarian injury caused a prompt decrease in AMH level (P < 0.01) and a further long-term decline in serum AMH (P = 0.017), indicating damage to the ovarian reserve. Pretreatment with GnRH-a diminished AMH-decrease (P < 0.05) and maintained serum AMH level in the long run (P < 0.05). Doxorubicin-exerted ovarian-vascular-injury is also displayed by an acute increase in ovarian VEGF level (P < 0.05) and a sustained decrease in serum AMH level (P < 0.001). This was followed by ovarian recovery manifested by increased neovascularization. GnRH-a delayed the recovery in AMH level and decreased the level of VEGF (P < 0.001), thus interfering with the vascular recovery subsequent to doxorubicin-induced vascular damage. LIMITATIONS, REASONS FOR CAUTION: To portray the differential mechanism of each chemotherapy, cyclophosphamide and doxorubicin were given separately, whereas most of the clinical protocols include several types of chemotherapies. Thus, future study should explore a prospective evaluation of various chemotherapies, as well as combined chemotherapeutic protocols. WIDER IMPLICATIONS OF THE FINDINGS: Our study demonstrates that different chemotherapy agents affect the ovary via diverse mechanisms and thus the administration of GnRH-a concomitantly, could be beneficial to a subpopulation of patients treated with cyclophosphamide-based protocols. STUDY FUNDING/COMPETING INTERESTS: This work was partially supported by a grant from the Israel Science Foundation (ISF) to I.B.-A. The authors have no conflict of interest to disclose.

Sensitization of gram-negative bacteria by targeting the membrane potential.

Toward generating new tools for fighting multidrug-resistant (MDR) bacteria, we assessed the ability of a membrane-active peptide to sensitize gram-negative bacteria to various antibiotics. The mechanism for affecting inner and/or outer membrane functions was assessed by complementary biophysical methods (SPR, DSC, ITC). The implication of efflux pumps was examined using Acr-AB mutants, as tested with representative antibiotics, host defense peptides, and synthetic mimics. The ability to affect disease course systemically was compared for a single therapy and combination therapy, using the mouse thigh-infection model. The data show that potent antibiotic action can be provoked in vitro and in vivo, by a treatment combining two antibacterial compounds whose individual inefficiency against gram-negative bacteria stems from their efflux. Thus, at subminimal inhibitory concentrations, the lipopeptide-like sequence, N(α)(ω7)dodecenoyl-lysyl-[lysyl-aminododecanoyl-lysyl]-amide (designated C12(ω7)K-ß12), has, nonetheless, rapidly achieved a transient membrane depolarization, which deprived bacteria of the proton-motive force required for active efflux. Consequently, bacteria became significantly sensitive to intracellular targeting antibiotics. Collectively, these findings suggest a potentially useful approach for expanding the antibiotics sensitivity spectrum of MDR gram-negative bacteria to include efflux substrates.

Antibacterial properties of an oligo-acyl-lysyl hexamer targeting Gram-negative species.

Toward developing new tools for fighting resistance to antibiotics, we investigated the antibacterial properties of a new decanoyl-based oligo-acyl-lysyl (OAK) hexamer, aminododecanoyl-lysyl-[aminodecanoyl-lysyl](5) (α(12)-5α(10)). The OAK exhibited preferential activity against Gram-negative bacteria (GNB), as determined using 36 strains, including diverse species, with an MIC(90) of 6.2 µM. The OAK's bactericidal mode of action was associated with rapid membrane depolarization and cell permeabilization, suggesting that the inner membrane was the primary target, whereas the observed binding affinity to lipoteichoic acid suggested that inefficacy against Gram-positive species resulted from a cell wall interaction preventing α(12)-5α(10) from reaching internal targets. Interestingly, perturbation of the inner membrane structure and function was preserved at sub-MIC values. This prompted us to assess the OAK's effect on the proton motive force-dependent efflux pump AcrAB-TolC, implicated in the low sensitivity of GNB to various antibiotics, including erythromycin. We found that under sub-MIC conditions, wild-type Escherichia coli was significantly more sensitive to erythromycin (the MIC dropped by >10-fold), unlike its acr-deletion mutant. Collectively, the data suggest a useful approach for treating GNB infections through overcoming antibiotic efflux.

Sensitization of Gram-negative bacteria to rifampin and OAK combinations.

While individually inefficient against Gram-negative bacteria, in-vitro combinations of rifampin and OAK were mutually synergistic since sub-minimal inhibitory concentrations of one compound have potentiated the other by 2-4 orders of magnitude. Synergy persisted in-vivo as single-dose systemic treatment of Klebsiella infected mice resulted in 10-20% versus 60% survival, respectively accomplished by individual and combined compounds. This outcome was achieved without drug formulation, rather, pharmacokinetic considerations have inspired the therapeutic regimen.

Pigment Epithelium-Derived Factor Alleviates Tamoxifen-Induced Endometrial Hyperplasia.

Tamoxifen is a cornerstone component of adjuvant endocrine therapy for patients with hormone-receptor-positive breast cancer. Its significant adverse effects include uterine hyperplasia, polyps, and increased risk of endometrial cancer. However, the underlying molecular mechanism remains unclear. Excessive angiogenesis, a hallmark of tumorigenesis, is a result of disrupted balance between pro- and anti-angiogenic factors. VEGF is a pro-angiogenic factor shown to be elevated by tamoxifen in the uterus. Pigment epithelium-derived factor (PEDF) is a potent anti-angiogenic factor that suppresses strong pro-angiogenic factors, such as VEGF. Our aim was to investigate whether angiogenic balance plays a role in tamoxifen-induced uterine pathologies, elucidate the molecular impairment in that network, and explore potential intervention to offset the proposed imbalance elicited by tamoxifen. Using in vivo mouse models, we demonstrated that tamoxifen induced a dose-dependent shift in endogenous uterine angiogenic balance favoring VEGF over PEDF. Treatment with recombinant PEDF (rPEDF) abrogated tamoxifen-induced uterine hyperplasia and VEGF elevation, resulting in reduction of blood vessels density. Exploring the molecular mechanism revealed that tamoxifen promoted survival and malignant transformation pathways, whereas rPEDF treatment prevents these changes. Activation of survival pathways was decreased, demonstrated by reduction in AKT phosphorylation concomitant with elevation in JNK phosphorylation. Estrogen receptor-α and c-Myc oncoprotein levels were reduced. Our findings provide novel insight into the molecular mechanisms tamoxifen induces in the uterus, which may become the precursor events of subsequent endometrial hyperplasia and cancer. We demonstrate that rPEDF may serve as a useful intervention to alleviate the risk of tamoxifen-induced endometrial pathologies.

Hormonal regulation of pigment epithelium-derived factor (PEDF) expression in the endometrium.

Pigment epithelium-derived factor (PEDF) is highly expressed in the female reproductive system and is subjected to regulation by steroid hormones in the ovary. As the uterine endometrium exhibits morphological and functional changes in response to estrogen (E2) and progesterone (P4), we aimed at characterizing the expression of PEDF in this component of the female reproductive tract and further at exploring the hormonal regulation of its expression. We found that PEDF is expressed in human and mouse endometrium. We further showed that this expression is subjected to regulation by steroid hormones, both in vivo and in vitro, as follows: E2 decreased PEDF expression and P4 increased its levels. In human endometrial samples, PEDF levels were dynamically altered along the menstrual cycle; they were low at the proliferative and early secretory phases and significantly higher at the late secretory phase. The expression levels of PEDF were inversely correlated to that of vascular endothelial growth factor (VEGF). We also showed that PEDF receptor was expressed in the endometrium and that its stimulation reduced VEGF expression. Illustrating the pattern of PEDF expression during the menstrual cycle may contribute to our understanding of the endometrial complexity.