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Estradiol-BODIPY linked via an 8-carbon spacer chain and 19-nortestosterone- and testosterone-BODIPY linked via an ethynyl spacer group were evaluated for cell uptake in the breast cancer cell lines MCF-7 and MDA-MB-231 and prostate cancer cell lines PC-3 and LNCaP, as well as in normal dermal fibroblasts, using fluorescence microscopy. The highest level of internalization was observed with 11β-OMe-estradiol-BODIPY 2 and 7α-Me-19-nortestosterone-BODIPY 4 towards cells expressing their specific receptors. Blocking experiments showed changes in non-specific cell uptake in the cancer and normal cells, which likely reflect differences in the lipophilicity of the conjugates. The internalization of the conjugates was shown to be an energy-dependent process that is likely mediated by clathrin- and caveolae-endocytosis. Studies using 2D co-cultures of cancer cells and normal fibroblasts showed that the conjugates are more selective towards cancer cells. Cell viability assays showed that the conjugates are non-toxic for cancer and/or normal cells. Visible light irradiation of cells incubated with estradiol-BODIPYs 1 and 2 and 7α-Me-19-nortestosterone-BODIPY 4 induced cell death, suggesting their potential for use as PDT agents.

The chemistry of gem-dibromovinyl derivatives has undergone a renaissance through the application of palladium catalysis and has been applied to pyrrole substituted gem-dibromovinyl BODIPY. gem-Dibromovinyl BODIPYs (substituted at either the [Formula: see text]-position of 8-phenyl or the [Formula: see text]-position of the pyrrole rings) were studied for cross-coupling reactions using Sonogashira, Suzuki, Heck and Stille conditions, and with phosphonates and thiols. The assigned structures were supported by MS and 1H NMR, [Formula: see text]C NMR, X-ray diffraction analysis as well as optical spectroscopy. The conjugates were investigated for their absorption, fluorescence and solvatochromic properties in different solvents. Substitution at the [Formula: see text]-position of 8-phenyl derivatives of gem-diethynyl BODIPYs did not induce any shift in the absorption maximum, while the [Formula: see text]-position pyrrole substituted derivatives showed a red shift. Aromatic compounds gave larger red shifts as compared to the aliphatic substituted analogs.

The systematic X-ray structure analyses of the primary cholesterol sidechain autoxidation products cholesterol 25- and 20β(S)-hydroperoxide are presented and compared to cholesterol and 25-hydroxycholesterol. Intermolecular interactions in crystal structures of the molecules are revealed through Hirshfeld surface analysis and fingerprint plots. The magnitude of energy frameworks is presented by combining efficient calculations of intermolecular interaction energies with novel graphical representation.

Fluorescence and SPECT/PET imaging are powerful tools currently in use by the scientific community and receiving a great attention for the development of dual-modality imaging agents. BODIPYs are among the most promising candidates to be used for such functions due their excellent absorbance and fluorescence properties as well as their ease of radiolabeling without compromising their biological properties. In this manuscript we present an overview of BODIPY radiolabeling methods and their relevance to the development of multimodality agents.

In a completely randomized experimental design with two replicates, the impact of iso-energetic supplements with different protein levels on body and wool growth was studied in eighty crossbreed (Merino Dohne × Corriedale) castrated male lambs (24.5 ± 4.4 kg of body weight, BW, 4 months of age) in summer (103 days) for three years. Animals were randomly allotted to four treatments (n = 20). All lambs grazed native pastures (NP) and animals in three treatments were daily supplemented (2% BW) with an iso-energetic supplement of different CP levels: control (CON, non supplemented), 12% CP (12CP), 16% CP (16CP) and 20% CP (20CP). Supplemented lambs had higher final BW (36.0, 36.4 and 37.7 kg for 12, 16 and 20CP, respectively) than CON lambs (28.9 kg) (P

Cytochrome P450 46A1 (CYP46A1) or cholesterol-24-hydroxylase is responsible for cholesterol metabolism and homeostasis in the human brain. More recently its activity has been linked to brain function and disease. The anti-HIV drug efavirenz activates CYP46A1 at low drug levels while inhibiting the enzyme activity at the high dose used in clinical practice. Synthetic analogs and hydroxylated metabolites of efavirenz enhance CYP46A1 activity, with reduced unwanted enzyme inhibition at higher concentrations. These observations provide a platform for structural modifications of efavirenz to modulate CYP46A1 activity as a therapeutic target of brain disorders such as Alzheimer's disease, for which currently no treatment is available.

This review focuses on progress in the development of different approaches to the design of steroid ([Formula: see text] estrogens, androgens, cholesterol) conjugates with coordination assemblies of metalloporphyrins, phthalocyanines and related complexes. Porphyrins and phthalocyanines have received considerable attention due to their novel composition, intriguing spectroscopic, photophysical, and redox properties, and potential application in light-harvesting and optoelectronic devices. With the development of more efficient imaging and therapeutic applications, these bio-conjugates are evaluated as multimodality agents (PET, fluorescence imaging) to monitor the mechanism of action of biologically active components in living systems and as agents for molecular recognition, oxygen atom transfer and catalysis. The tetrapyrrole components, which can be coupled via covalent and various non-covalent linkages, may exhibit strong interactions through efficient photo-induced electron and/or energy transfer processes.

After encouraging preclinical and human dosimetry results for the novel estrogen receptor (ER) PET radiotracer 4-fluoro-11β-methoxy-16α-18F-fluoroestradiol (18F-4FMFES), a phase II clinical trial was initiated to compare the PET imaging diagnostic potential of 18F-4FMFES with that of 16α-18F-fluoroestradiol (18F-FES) in ER-positive (ER+) breast cancer patients. Methods: Patients diagnosed with ER+ breast cancer (n = 31) were recruited for this study, including 6 who underwent mastectomy or axillary node dissection. For each patient, 18F-FES and 18F-4FMFES PET/CT scans were done sequentially (within a week) and in random order. One hour after injection of either radiotracer, a head-to-thigh static scan with a 2-min acquisition per bed position was obtained. Blood samples were taken at different times after injection to assess each tracer metabolism by reverse-phase thin-layer chromatography. The SUVmean of nonspecific tissues and the SUVmax of the tumor were evaluated for each detected lesion, and tumor-to-nonspecific organ ratios were calculated. Results: Blood metabolite analysis 60 min after injection of the tracer showed a 2.5-fold increase in metabolic stability of 18F-4FMFES over 18F-FES. Although for most foci 18F-4FMFES PET had an SUVmax similar to that of 18F-FES PET, tumor contrast improved substantially in all cases. Lower uptake was consistently observed in nonspecific tissues for 18F-4FMFES, notably a 4-fold decrease in blood-pool activity as compared with 18F-FES. Consequently, image quality was considerably improved using 18F-4FMFES, with lower overall background activity. As a result, 18F-4FMFES successfully identified 9 more lesions than 18F-FES. Conclusion: This phase II study with ER+ breast cancer patients showed that 18F-4FMFES PET achieves a lower nonspecific signal and better tumor contrast than 18F-FES PET, resulting in improved diagnostic confidence and lower false-negative diagnoses.

To develop receptor based fluorescence ligands for imaging breast and prostate cancer, a series of estrogen-, testosterone- and 19-nortestosterone conjugates linked to BODIPY (4,4-difluoro-4-bora-3a,4a-diaza-s-indacene) or aza-BODIPY, were prepared. Their synthesis involves attachment of iodo derivatives of differently substituted BODIPY and aza-BODIPY analogs to the C17α-position of the steroid moieties using either the Sonogashira coupling or Click reaction. The UV-Vis absorption spectra of the conjugates range from 500 to 710nm with fluorescence emission properties ranging from 520 to 700nm, facilitating observations in living cells and tissues. Selection of the site of substitution, as well as the type of substituents on the steroidal moiety and the use of different linkers, provides a library of fluorescing conjugates to explore the effect of structural modifications on biological properties.

In vivo imaging of estrogen receptor (ER) densities in human breast cancer is a potential tool to stage disease, guide treatment protocols and follow-up on treatment outcome. Both positron emission tomography (PET) and fluorescence imaging have received ample attention to detect ligand-ER interaction. In this study we prepared BODIPY-estradiol conjugates using 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene (BODIPY) as fluorescent probe and estradiol derivatives as ligand and established their relative binding affinity (RBA) for the ERα. The synthesis of the conjugates involves attachment of a BODIPY moiety to the C17α-position of estradiol using Sonogashira or click reactions of iodo-BODIPY or aza-BODIPY with various 17α-ethynylestradiol (EE2) derivatives. The highest RBA for the ERα was observed with the EE2-BODIPY conjugate (7) featuring a linear eight carbon spacer chain. Cell uptake studies and in vivo imaging experiments in an ER-positive mouse tumor model are in progress.

This paper describes the morphosyntactic behavior of different semantic types of property words in a balanced sample of 36 Oceanic languages. After a brief general introduction to the functional typology of property words, I first discuss diversity in Oceanic property word classes from a family-internal perspective. In the second part of the paper, Oceanic property words are placed in a world-wide typological perspective. Specifically, I test their behavior with regard to two implicational universals proposed in the literature, concerning the relation between the encoding of predicative property words, the presence of grammatical tense, and locus of marking at the clause level. In typological studies, the Oceanic language family has been claimed to display verbal predicative property words, to lack tense, and to be head- or zero-marking, with marginal exceptions. This paper shows that, even though such an overall profile can be discerned, Oceanic property words exhibit more variation than is acknowledged in crosslinguistic research. Moreover, my findings for property word classes are fitted into a larger picture of lexical categorization in Oceanic languages.

In 1990, two selection lines of Merino sheep were established for low and high behavioural reactivity (calm and nervous temperament) at the University of Western Australia. Breeding records consistently showed that calm ewes weaned 10% to 19% more lambs than the nervous ewes. We hypothesise that calm ewes could have a higher ovulation rate than nervous ewes and/or calm ewes could have a lower rate of embryo mortality than nervous ewes. We tested these hypotheses by comparing the ovulation rate and the rate of embryo mortality between the calm and nervous lines before and after synchronisation and artificial insemination. Merino ewes from the temperament selection lines (calm, n=100; nervous, n=100) were synchronised (early breeding season) for artificial insemination (day 0) (intravaginal sponges containing fluogestone acetate and eCG immediately after sponge withdrawal). On day-17 and 11 ovarian cyclicity and corpora lutea, and on days 30 and 74 pregnancies and embryos/foetuses were determined by ultrasound. Progesterone, insulin and leptin concentrations were determined in blood plasma samples from days 5, 12 and 17. Ovarian cyclicity before and after oestrus synchronisation did not differ between the lines, but ovulation rate did (day-17: calm 1.63; nervous 1.26; P

A family of new asymmetric and symmetric 1,3,7,9-tetramethyl-4,4-bora difluoro-diaza-s-indacene (BODIPY) derivatives, bearing gem-dibromovinyl substituents, was synthesized by the Corey-Fuchs olefination method. One or two gem-dibromovinyl moieties were attached at either the p-position of 5-phenyl, or the β-position of the pyrrole ring, directly or, through phenyl spacers. The assigned structures were supported by MS, NMR (1H, 13C, 19F), X-ray diffraction analysis and for some compounds 2D HSQC and 11B NMR as well as optical spectroscopy. Their absorption and fluorescence properties and solvatochromism in different solvents were investigated. The highest absorption and emission maxima were obtained for compounds having two gem-dibromovinyl groups attached directly or through the phenyl spacer. The best correlation (R-coefficient) between the solvent and spectral properties of the BODIPYs were obtained using the refractive index of the solvent. Although these compounds are structurally quite similar, their solid states show remarkable differences in the crystal system, clearly revealing two distinct patterns of gem-dibromovinyl orientation and torsion angles of the 5-phenyl ring and the indacene plane. Hirshfeld surface analysis data were used to visualize various intermolecular interactions.

Steroids linked to porphyrin fluorophores are being developed to improve the selectivity of photosensitizers for photodyanamic therapy and fluorescence imaging of steroid-hormone positive cancers. A number of porphyrin derivatives substituted with one or two steroid moieties coupled via a 17[Formula: see text]-ethynyl group of estradiol, testosterone and 19-nortestosterone were synthesized using Pd(II) as a catalyst.

The design of antibody-conjugates (ACs) for delivering molecules for targeted applications in humans has sufficiently progressed to demonstrate clinical efficacy in certain malignancies and reduced systemic toxicity that occurs with standard nontargeted therapies. One area that can advance clinical success for ACs will be to increase their intracellular accumulation. However, entrapment and degradation in the endosomal-lysosomal pathway, on which ACs are reliant for the depositing of their molecular payload inside target cells, leads to reduced intracellular accumulation. Innovative approaches that can manipulate this pathway may provide a strategy for increasing accumulation. We hypothesized that escape from entrapment inside the endosomal-lysosomal pathway and redirected trafficking to the nucleus could be an effective approach to increase intracellular AC accumulation in target cells. Cholic acid (ChAc) was coupled to the peptide CGYGPKKKRKVGG containing the nuclear localization sequence (NLS) from SV-40 large T-antigen, which is termed ChAcNLS. ChAcNLS was conjugated to the mAb 7G3 (7G3-ChAcNLS), which has nanomolar affinity for the cell-surface leukemic antigen interleukin-3 receptor-α (IL-3Rα). Our aim was to determine whether 7G3-ChAcNLS increased intracellular accumulation while retaining nanomolar affinity and IL-3Rα-positive cell selectivity. Competition ELISA and cell treatment assays were performed. Cell fractionation, confocal microscopy, flow cytometry, and Western blot techniques were used to determine the level of antibody accumulation inside cells and in corresponding nuclei. In addition, the radioisotope copper-64 ((64)Cu) was also utilized as a surrogate molecular cargo to evaluate nuclear and intracellular accumulation by radioactivity counting. 7G3-ChAcNLS effectively escaped endosome entrapment and degradation resulting in a unique intracellular distribution pattern. mAb modification with ChAcNLS maintained 7G3 nM affinity and produced high selectivity for IL-3Rα-positive cells. In contrast, 7G3 ACs with the ability to either escape endosome entrapment or traffic to the nucleus was not superior to 7G3-ChAcNLS for increasing intracellular accumulation. Transportation of (64)Cu when complexed to 7G3-ChAcNLS also resulted in increased nuclear and intracellular radioactivity accumulation. Thus, ChAcNLS is a novel mAb functionalizing technology that demonstrates its ability to increase AC intracellular accumulation in target cells through escaping endosome entrapment coupled to nuclear trafficking.

BODIPY and aza-BODIPY fluorophores linked to steroids are being developed as multimodal-imaging agents to monitor the mechanism of action of biologically active components in living systems.

A series of BODIPYs (boron-dipyrromethene dyes; 4,4-difluoro-5-aryl-4-bora-3a,4a-diaza-s-indacene) derivatives, bearing an aldehyde group at different pyrrole positions, were transformed into gem-dibromovinyl analogs using the Corey-Fuchs or Lautens olefination method. The gem-dibromovinyl moieties (one or two) are symmetric/asymmetric and attached at α- or β-positions of the pyrrole ring, directly or through the extension of a β-position substituted phenyl spacer ring. The molecular structures of the resultant dyes were assigned using MS, 1H, 13C, 19F NMR, X-ray diffraction and for some compounds 2D HSQC and 11B NMR. The absorption, fluorescence and solvatochromism properties were investigated in different solvents. The highest absorption and emission maxima were obtained for compounds having two gem-dibromovinyl groups attached directly at the α-positions. The best correlation (R-coefficient) of absorption between the solvents and spectral properties of the BODIPYs was obtained using the refractive index of the solvent. The R-coefficient of emission was obtained only for compounds having gem-dibromo vinyl moieties at α-positions. Their solid states clearly reveal distinct patterns of gem-dibromovinyl orientation, torsion angles of the 5-phenyl ring and the indacene plane. Hirshfeld surface analyses were used to visualize various intermolecular interactions.

The effect iso-energetic supplements (2.9 Mcal/kg DM; 2 % body weight as-dry fed basis) with different crude protein (CP) levels (12, 16 and 20 % CP) during early post-weaning phase were evaluated on lamb carcass and meat quality traits. For three years (January–April; 2013, 2015 and 2016), eighty crossbreed lambs (Dohne Merino × Corriedale, with an initial average body weight -BW- of 24.5 ± 4.4 kg with 4 months of age) were randomly allotted each year to the following treatments: CON: native pastures (NP) without S (supplement); 12CP: NP + S with 12 % CP; 16CP: NP + S with 16 % CP; 20CP: NP + S with 20 % CP. Thereafter (April-July), all animals were managed together on an annual forage winter crop (ryegrass or oat) without supplementation until an average slaughter weight of 43 kg was reached. Supplementation with different crude protein (CP) levels for approximately 3 months during an early post-weaning rearing period in lambs grazing native pastures, resulted in general in greater pre-slaughter body weight, carcass weight and compactness index without significant effects (P > 0.05) on carcass high-value cuts, meat color parameters, pH, and Warner-Bratzler shear force (WBSF) values after a common feeding period on improved pastures. The beneficial fatty acids ratio (n6/n3) linked with human health favoured those animals fed exclusively on pastures. In general, among supplemented groups, the magnitude of the changes found were of little relevance.

Summary Purpose To monitor a real-time follow-up of tumor response to photodynamic therapy (PDT) by dynamic 2-deoxy-2-[ 18 F]fluoro- d -glucose ( 18 FDG) and positron emission tomography (PET) using two photosensitizing drugs in vivo, and to assess their mechanisms of action. Methods Two types of photosensitizers with different action mechanisms were used in rats implanted with two tumors: AlPcS4 mainly affecting the tumor vascular system, and ZnPcS2 largely inducing direct cell kill. Twenty-four hours after administration of either photosensitizer, one tumor served as control while the other was treated with red light during 30 min within the 2 h PET imaging by infusion of 18 FDG. The usual two-tissue compartment kinetic model was modified to take into account the perturbation of the treatment during imaging. Results The illumination of the tumors during PET imaging provoked a net decrease of 18 FDG uptake in tumors treated with AlPcS4 and a near total absence of 18 FDG uptake in tumors treated with ZnPcS2. After the end of illumination, the tumors regained 18 FDG uptake with a more pronounced uptake in the tumors treated with ZnPcS2. The rate constant values of the new 18 FDG kinetic model reflected the response of the tumors to the treatment in both photosensitizers. Conclusions Dynamic PET imaging can be used to quantitatively assess in vivo and in real-time the response of tumors to treatments. It is demonstrated that the 30 min of treatment was not sufficient to reduce the activity of the tumors. The technique could be extended to directly monitor the effects of drugs in vivo.

The interaction of primary autoxidation products of cholesterol, 25- and 20ξ-hydroperoxides, with the four principal cholesterol-metabolizing cytochrome P450 enzymes is reported. Addition of cholesterol 25-hydroperoxide to CYP27A1 and CYP11A1 induced well-defined spectral changes while generating 25-hydroxycholesterol as major product along with small amounts of triols. The 20ξ-hydroperoxides induced spectral shifts in CYP27A1 and CYP11A1, yet glycol metabolites were detected only with CYP11A1. CYP7A1 and CYP46A1 failed to give metabolites with any of the hydroperoxides. A P450 hydroperoxide-shunt reaction is proposed, where the hydroperoxides serve both as donor for reduced oxygen and as substrate. For the first time, CYP27A1 is shown to mediate the reduction of cholesterol 25-hydroperoxide to 25-hydroxycholesterol, a role of potential significance for cholesterol-rich tissues with high oxidative stress. CYP27A1 may participate in these tissues in removal of harmful autoxidation products, while providing a complementary source for 25-hydroxycholesterol, a modulator of immune cell function and mediator of viral cell entry.

Cyclotron production of 99mTc is a promising route to supply 99mTc radiopharmaceuticals. Higher 99mTc yields can be obtained with medium-energy cyclotrons in comparison to those dedicated to PET isotope production. To take advantage of this capability, evaluation of the radioisotopic purity of 99mTc produced at medium energy (20–24 MeV) and its impact on image quality and dosimetry was required. Methods: Thick 100Mo (99.03% and 99.815%) targets were irradiated with incident energies of 20, 22, and 24 MeV for 2 or 6 h. The targets were processed to recover an effective thickness corresponding to approximately 5-MeV energy loss, and the resulting sodium pertechnetate 99mTc was assayed for chemical, radiochemical, and radionuclidic purity. Radioisotopic content in final formulation was quantified using γ-ray spectrometry. The internal radiation dose for 99mTc-pertechnetate was calculated on the basis of experimentally measured values and biokinetic data in humans. Planar and SPECT imaging were performed using thin capillary and water-filled Jaszczak phantoms. Results: Extracted sodium pertechnetate 99mTc met all provisional quality standards. The formulated solution for injection had a pH of 5.0−5.5, contained greater than 98% of radioactivity in the form of pertechnetate ion, and was stable for at least 24 h after formulation. Radioisotopic purity of 99mTc produced with 99.03% enriched 100Mo was greater than 99.0% decay corrected to the end of bombardment (EOB). The radioisotopic purity of 99mTc produced with 99.815% enriched 100Mo was 99.98% or greater (decay corrected to the EOB). The estimated dose increase relative to 99mTc without any radionuclidic impurities was below 10% for sodium pertechnetate 99mTc produced from 99.03% 100Mo if injected up to 6 h after the EOB. For 99.815% 100Mo, the increase in effective dose was less than 2% at 6 h after the EOB and less than 4% at 15 h after the EOB when the target was irradiated at an incident energy of 24 MeV. Image spatial resolution and contrast with cyclotron-produced 99mTc were equivalent to those obtained with 99mTc eluted from a conventional generator. Conclusion: Clinical-grade sodium pertechnetate 99mTc was produced with a cyclotron at medium energies. Quality control procedures and release specifications were drafted as part of a clinical trial application that received approval from Health Canada. The results of this work are intended to contribute to establishing a regulatory framework for using cyclotron-produced 99mTc in routine clinical practice.

Various zinc phthalocyanines were substituted at ortho or meta positions with 1,3,5,7-tetramethyl-8-phenyl-4,4-difluoroboradiazaindacene (BODIPY) moieties via Pd catalyzed Sonogashira-coupling reactions, at either meso or β positions of the BODIPY. UV–Vis and fluorescence emission spectra of the conjugates exhibit panchromatic behavior due to their absorption over a broad spectral region from ultraviolet to visible and energy transfer from the excited BODIPY substituent to the phthalocyanine core. Some conjugates show red-shifted Q-bands that correlate to the planarity and dihedral angle between the subunits, as well as the position and number of attached BODIPY and phthalocyanine moieties.

Temperament can be defined as the fearfulness and reactivity of an animal in response to humans and strange, novel or threatening environments. The productive performance of an animal is affected by its temperament, and selection of calm animals might improve their adaptation to the farming environment and handling, as well as improve productivity. The temperament was measured in lambs of two breeds of sheep in Uruguay. The effects of dam’s age, type of birth, age of the lamb and contemporary group (CG; lambs belonging to the same year, flock, sex and rearing group) on the temperament of the lambs and the heritability of temperament were estimated with a Bayesian analysis using Gibbs sampling. Overall, 4962 Corriedale lambs and 2952 Merino lambs from 13 farms were tested. Temperament was measured using the isolation box test, isolating a lamb inside the box for 30 s, and recording the vibrations produced by its movements. The average temperament score (±s.e.m.) of the Corriedale lambs was 24.7 (±0.23) and that of the Merino was 36.8 (±0.45). Temperament was not associated with dam’s age, type of birth or lamb’s age. There were no relevant differences in the agitation score between lambs born in 2010 and 2011. The mean of the distribution of possible values of heritability (±s.d.) was 0.18 (±0.05) for the Corriedale and 0.31 (±0.06) for the Merino. The likelihood of heritability values to be greater than 0.15 exceeded 70% in the Corriedale and 90% in the Merino. The temperament of Merino and Corriedale sheep in Uruguay is moderately heritable. It is not related to dam’s age, type of birth or age of the lambs; however, it is affected by some aspect of the CG.

Background: The current article explores the associations between home gardening and dietary behaviors, physical activity, mental health, and social relationships among secondary school students in New Zealand.Method: Data were drawn from a national youth health and well-being survey, conducted in 2012. In total, 8,500 randomly selected students from 91 randomly selected secondary schools completed the survey.Results: Two thirds of students had a vegetable garden at home and one quarter of all students participated in home gardening. Students participating in gardening were most likely to be male, of a Pacific Island ethnicity, of younger age, and living in a rural area. Gardening was positively associated with healthy dietary habits among students, such as greater fruit and vegetable consumption. Gardening was also positively associated with physical activity and improved mental health and well-being. Students who participate in gardening report slightly lower levels of depressive symptoms and enhanced emotional well-being and experience higher family connection than students who do not participate in gardening.Conclusions: Gardening may make a difference for health and nutrition behaviors and may contribute to adolescents’ health and well-being in a positive manner. Health promoters should be encouraged to include gardening in future interventions for young people.

Methods for the preparation of a variety of covalently linked Pc–Pc homo-dimers (ortho and meta), heterodimers and Pc–Pc triads linked through NH and bridged viaN-aryl (phenyl) groups were developed. The palladium catalyzed intermolecular carbon–nitrogen coupling reaction (Buchwald–Hartwig) between Pc-iodo and Pc-amino phthalocyanine derivatives was used for these studies. Photophysical data reveal energy transfer between the Pc moieties resulting in the appearance of new red-shift Q-bands that correlate to the planarity and dihedral angle between the subunits. The shift and the nature of Q-band depend on the number of phenyl groups, the number of Pc and the position of attachment on the phenyl ring.

Several phthalocyanine dimers, trimers, and oligomers bridged via aryl (phenyl) groups were prepared using the Suzuki–Miyaura cross coupling reaction of phthalocyanine-boronate ester and various halide derivatives under palladium catalyst reaction conditions. Photophysical data reveal energy transfer between the Pc moieties resulting in the appearance of new red-shifted Q-bands. The shift and the nature of Q-band depend on the number of phenyl groups, the number of Pc, and the position of attachment on the phenyl ring.

There is strong evidence that the gonads modulate the hypothalamic–pituitary–adrenal axis. To investigate these sex differences at the adrenal glands of sheep we compared the cortisol response to ACTH (experiment 1) and measured the relative expression of oestrogen receptor alpha (ERS1), androgen receptor (AR), melanocortin 2 receptor (MC2R) and steroid acute regulatory protein (STAR) mRNA in adrenal glands (experiment 2) of gonadectomised rams and ewes either with or without sex steroid replacement. In experiment 1 six castrated adult rams and four ovariectomised adult ewes were used in two ACTH trials. On each trial blood samples were taken every 15 min for 4 h through an indwelling jugular catheter and each animal received 0.5 mg of an ACTH analogue i.v., immediately after the sample at 1 h from the beginning of the trial. Four days after the first trial the males received 100 mg of Testosterone Cyclopentilpropionate (TC) i.m. and the females received 2.5 mg of Oestradiol Benzoate (EB) i.m. At 72 h after TC or EB administration the second trial was performed. In experiment 2 the adrenal glands were obtained from gonadectomised adult rams (n=8) and adult ewes (n=8). Four rams received 100 mg of TC i.m. and four females received 0.5 mg of EB i.m. Blood samples were taken at 0, 12, 24, 48 and 72 h relative to steroid replacement and the animals were thereafter slaughtered. Cortisol, testosterone and 17β-oestradiol were determined by radioimmunoanalysis. The transcripts of ERS1, AR, MC2R and STAR were determined by real-time reverse transcription PCR in adrenal tissue. Cortisol secretion was higher in female sheep than in male sheep, and higher in EB-treated than non-treated ewes. No difference in cortisol secretion was observed between TC-treated and non-treated rams. Gonadectomised rams treated with TC presented greater AR mRNA and MC2R mRNA expression than males without the steroid replacement. Gonadectomised ewes treated with EB tended to present lower AR mRNA than the ones without steroid replacement. Gonadectomised rams with TC also had greater AR mRNA, ERS1 mRNA and MC2R mRNA expression than ewes treated with EB. The relative amount of STAR transcript was not different among the different groups. The results confirm sex differences in ACTH-induced cortisol secretion in sheep, as well as in the expression of the receptor proteins for both 17β-oestradiol and testosterone in the sheep adrenal gland. However, the underlying mechanisms for sex steroid modulation remain unresolved.

The synthesis of a series of new fluorinated phthalocyanines and porphyrins as potential bimodal probes is evaluated. In these complexes, zinc phthalocyanine functions as a fluorescence imaging moiety while attachment of an aliphatic chain of different lengths bearing a tosylate group permits introduction of fluorine via nucleophilic substitution of the tosylate group. Using short-lived [18F]fluoride gives the analogous 18 F -radiolabelled tracer rendering the bimodal probe suitable for both fluorescence and PET imaging.

Rhodium-catalyzed carbon–carbon bond forming reactions using phthalocyanine-boronate ester were investigated. The reaction of Pc-boronate ester with substituted aldehydes gave 1,2-addition products (secondary alcohols), while α,β-unsaturated carbonyl compounds (aldehydes, ketones, esters, and amides) furnished 1,4-addition products. The reaction of Pc-substituted alkynes with o -formyl and acetyl phenylboronic acid gave 1 H - and 1-methyl inden-1-ol annulation derivatives. All reactions were performed in mixtures of organic–aqueous solvents.

The aim of this study was to compare the in vivo stability, uptake, and positron emission tomography (PET) imaging performance of a novel estrogen receptor PET tracer, 4-fluoro-11β-methoxy-16α-[(18)F]fluoroestradiol (4FMFES), with 16α-[(18)F]fluoroestradiol (FES).MC7-L1 and MC4-L2 (ER+) cell lines and their ERα-knockdown variants (ERαKD) were implanted subcutaneously in Balb/c mice. After 21 days, mice were imaged using either FES or 4FMFES. One hour post-injection, static images were acquired for 30 min and the tumor %ID/g uptake values were derived. Biodistribution data were also obtained 1 h following the injection of either FES or 4FMFES. Blood samples were taken at different times and analyzed on thin-layer chromatography to quantify the presence of radiometabolites for each radiotracer. To assess specific targeting to the estrogen receptors, mice bearing only ER+ tumors were treated with the competitive ER inhibitor fulvestrant 48 h prior to imaging with 4FMFES.Metabolic stability was found to be similar for both tracers in mice. Both FES and 4FMFES differentiated ER+ tumors from ERαKD tumors in biodistribution and PET imaging studies. 4FMFES achieved a significantly higher %ID/g uptake in ER+ tumors and MC4-L2 ERαKD tumors than FES in the PET imaging studies. Also, tumor-to-background ratio was higher in ER+ tumors using 4FMFES compared to FES. Dissection data showed a significantly higher %ID/g in all tested cell lines and ER-rich tissues using 4FMFES versus FES. Fulvestrant-treated mice had either low or undetectable tumor uptake.In a tumor-bearing mouse model, 4FMFES achieves better specific tumor uptake and better contrast than FES, making it a promising candidate for ER imaging.

The synthesis of a series of new zinc phthalocyanine-peptide conjugates targeting the gastrin-releasing peptide (GRP) and integrin receptors is reported. Two alternative synthetic methods based on Sonogashira cross-coupling of an iodinated zinc phthalocyanine with acetylenic bombesin or arginine-glycine-aspartic acid (RGD) derivatives, either in solution or on solid phase, are presented. The water-soluble conjugates were screened for their photodynamic efficacy against several cancer cell lines expressing different levels of GRP and integrin receptors, and their intracellular localization was evaluated via confocal fluorescence microscopy. Variations in photocytotoxicity between the conjugates correlate to differences in hydrophobicity as well as receptor-mediated cell uptake. In the case of the phthalocyanine-bombesin conjugate, competition experiments confirm the involvement of the GRP receptor in both the phototherapeutic activity as well as intracellular localization. These findings warrant further in vivo studies to evaluate the potential of this conjugate as photosensitizer for photodynamic therapy (PDT) of cancers overexpressing the GRP receptor.

Contents The aims were to evaluate sperm DNA fragmentation (SDF) in boars through the dispersion of their chromatin in raw semen samples, quantifying the extent of SDF, and to assess dynamic aspects of sperm DNA damage after incubation to obtain the rate of sperm DNA fragmentation (rSDF) under thermal conditions similar to the uterus (37°C) over a period of up to 24 hr and to correlate the reproductive outcome of the sows with the SDF of the boars at ejaculation. The study was performed on a pig-breeding farm in southern Uruguay. Sixty-one ejaculates from five of the most frequently used hybrid boars were evaluated. Semen was collected weekly from each of the boars, using the gloved-hand technique and discarding the jelly-like fraction of the ejaculate. Fresh semen was kept in a water bath at 37°C and protected from light, and was thereafter processed with Sperm-Sus-Halomax® to evaluate SDF. The smears for time 0 (T0) were made on farm, and thereafter smears were made at the laboratory at 4 hr of obtaining the semen (T4), then every 2 hr (T6, T8, T10, T12) and a final fixation at 24 hr (T24). Differences in SDF were observed among exposure times for all boars (p

Dynamic positron emission tomography (PET) combined with the constant infusion of 2-deoxy-2-[(18)F]fluoro-D-glucose (FDG) as a tracer permits real-time monitoring of systemic transient metabolic changes resulting from photodynamic therapy (PDT) in tumour bearing animals. The effect of PDT on tumour FDG uptake rates was evaluated using four different sulfonated phthalocyanine analogs as photosensitizers (PS) in combination with either continuous or fractionated illumination protocols. Mice bearing two EMT-6 tumours were infused with FDG to start PDT 30 min later. Dynamic images were acquired to produce FDG uptake over time for the treated and reference tumours. Practically all PDT protocols induced a reduction in the FDG uptake rates in the treated tumour during PDT, except for the zinc tetrasulfophthalocyanine, when using fractionated light, reflecting the low photodynamic efficacy of this PS. In general, the response to PDT was characterized by a rebound in the FDG uptake rate after illumination. A strong drop in FDG tumour uptake rates during PDT, followed by a strong rebound, together with short delay-to-response times, corresponded to optimal long-term tumour response outcomes. This dynamic FDG-PET protocol provides real-time observations to predict long-term PDT efficacy, while using fewer animals than conventional methods, thus making possible the rapid optimization of treatment parameters.

An easy synthetic method for the preparation of pyrazine-2,3-dicarbonitrile 5,6-bis-substituted derivatives using Pd-catalyzed cross coupling reaction (Sonogashira, Suzuki, Heck) is described. The reaction conditions allow preparing symmetrical and unsymmetrical bis-substituted products in moderate to high yields, as precursors for the preparation of highly substituted symmetrical and non-symmetrical aza-phthalocyanines with well-defined spectral properties.

This paper is a corpus-based study of non-prototypical ditransitive constructions with two highly salient - because human - non-agentive arguments. We evaluate claims made in earlier studies about this type of construction, using data from English, Dutch, and Polish corpora. Our study confirms that ditransitives with two human non-agents are (very) infrequent in discourse, and - depending on the argument coding strategy of a specific language - may involve ambiguity concerning the assignment of the Theme and Recipient roles. These characteristics are reflected in the formal expression of these constructions across languages. In addition, our data shows the effects of individual lexical verbs in determining frequency and expression patterns of non-prototypical ditransitives.

Sulfonated metallo phthalocyanines (MPcS(n)) are second generation photosensitizers advanced for photodynamic therapy of various medical applications. A series of ZnPcS(n) was demetallated and subsequently converted to the corresponding [(64)Cu]CuPcS(n) in 40-50% isolated yields and >98% radiochemical purities. Tumor-bearing mice were injected with the (64)Cu-labeled products and subjected to 3-h dynamic PET imaging studies. Biodistribution patterns showed characteristic differences between the various derivatives. Tumor uptake was detected only for the amphiphilic derivatives [(64)Cu]CuPcS(2) and [(64)Cu]CuPcS(3)C(6) (1-1.5%ID/g). The biological data suggest that PET imaging with [(64)Cu]CuPc can be used to establish structure-PDT efficacy relationships for Pc-based photosensitizers.

The reaction of Ni (II) complexes of etioporphyrins I, octaethylporphyrins, coproporphyrinsI tetramethylester, methyl pyrroporphyrin XXI ethylester and mesoporphyrin IX dimethyl ester derivatives with N-bromosuccinimide and triethylamine trihydrofluoride gave mono- through tetra-meso-brominated derivatives as the principal products. β-brominated products were also detected with the simultaneous loss of the β-alkyl substituents. Reaction of octaethylporphyrin Cu (II) was slow yielding meso-mono and dibromo derivatives as major products. Prolongation of the reaction time resulted in the formation of tri- and tetrabromo products substituted at both meso- and β-positions. Our results show that the central metal ion plays a major role in these bromination reactions.

Phthalocyanines (Pc) were conjugated with peptide moieties to improve their target selectivity for potential use as fluorescence and/or positron emission tomography (PET) probes in medical imaging. Three synthetic methods based on palladium-catalyzed cross-coupling reactions (Sonogashira, Buchwald-Hartwig, and Suzuki-Miyaura) were investigated. Using these methods, a series of peptides monofunctionalized with Pc at the N/C-terminal position or on a phenylalanine side chain was obtained in good yields and characterized.

Two cationic porphyrins bearing an isothiocyanate group for conjugation to monocolonal antibodies have been synthesized. The two porphyrins conjugated efficiently to three monoclonal antibodies (anti-CD104, anti-CD146 and anti-CD326), which recognize antigens commonly over-expressed on a range of tumour cells. In vitro, all conjugates retained the phototoxicity of the porphyrin and the immunoreactivity of the antibody. Mechanistic studies showed that conjugates formed from the mono- and tri-cationic porphyrin and anti-CD104 antibody mediated apoptosis following irradiation with non-thermal red light of 630 ± 15 nm wavelength. In vivo antibody conjugates caused suppression of human LoVo tumour growth in immunodeficient NIH III mice, similar to the commercial photodynamic therapy (PDT) agent Photofrin, but at administered photosensitizer doses that were more than two orders of magnitude lower. Positron emission tomography (PET) following PDT showed a large, early increase in uptake of (18) fluorodeoxyglucose (FDG) by tumours treated with the anti-CD104 conjugates. This effect was not observed with Photofrin or with conjugates formed from the same photosensitizers conjugated to an irrelevant antibody.

Four experiments were conducted to evaluate the effectiveness of a new controlled drug releasing device containing 0.3 g progesterone (DICO ® ) on ovarian control in sheep. In experiment 1, serum progesterone concentrations induced by a 14 days treatment of DICO ® ( n = 9) and CIDR-G ® ( n = 9) were compared in ovariectomized ewes. Both devices induced similar responses and no differences were recorded. In experiment 2, the onset of oestrus and the time of ovulation obtained after 14 days treatment with DICO ® ( n = 8) and CIDR-G ® ( n = 7) were compared in cyclic ewes. Both devices induced oestrus and ovulation in all of the ewes. The onset of oestrus (34.5 ± 2.8 and 30.0 ± 7.7 h), the time of ovulation (60.0 ± 9.1 and 54.9 ± 6.4 h), the ovulation rate (1.3 ± 0.5 and 1.4 ± 0.5), the follicular diameter at ovulation (7.0 ± 0.8 and 7.3 ± 1.1 mm), and the lifespan of the ovulatory follicles (8.6 ± 2.2 and 10.0 ± 2.9 days) were similar for the DICO ® and CIDR-G ® devices, respectively. In Experiment 3, the re-utilization of DICO ® devices inserted for 6 days (i.e. short-term protocol) was evaluated in ovariectomized ewes. The females received a re-used (previously used for 6 days; n = 11) or a new DICO ® ( n = 11) for a period of 6 days. The re-used DICO ® devices induced a lower serum progesterone concentration than the new devices ( P ® device maintained serum progesterone concentrations above 7.1 nmol/L (i.e. >2 ng/ml) throughout treatment. In Experiment 4, the administration of eCG treatment at DICO ® withdrawal was evaluated in cyclic ewes. The short-term protocol using DICO ® devices for 6 days was applied with ( n = 8) or without ( n = 7) 300 IU eCG at the time of device withdrawal. The administration of eCG advanced ovarian follicular development, synchronizing the onset of oestrus at 36 h and the time of ovulation at 60 h from device withdrawal. In conclusion, data from these experiments show the use of DICO ® or CIDR-G ® devices containing 0.3 g of progesterone to have a similar efficiency in controlling serum progesterone concentrations, follicular development and the time of ovulation in sheep. The re-use of the devices, associated with the short-term protocol for 6 days is possible, although further studies on induced fertility rates are warranted.

The impact of lipophilicity on biological parameters critical to photodynamic efficacy was analyzed for a new generation of trisulfobenzo(mononaphtho)porphyrazines. The porphyrazines were substituted on the naphtho ring with linear alkynyl side chains of various lengths. When compared to the analogous phthalocyanine structures, the added benzo ring in the porphyrazine structures increased the lipophilicity for analogs with short alkynyl chains, while this effect disappeared for analogs with longer side chains. In aqueous media, the analogous phthalocyanine series showed aggregation tendencies. In contrast, no correlation between aggregate formation and the length of the alkynyl side chain was evident in the porphyrazine series. At low concentrations, the length of the side chain did not affect cell uptake, while phototoxicity towards EMT-6 mouse tumour cells showed a parabolic relationship, where the hexynyl derivative showed the highest activity. The trisulfonated porphyrazines localized at intracellular organelles, plasma and perinuclear membranes, but could not be found in the nucleus. Total cell uptake of dye did not correlate with phototoxicity, suggesting that localization in certain intracellular organelles, and distribution into critical intracellular sites are important determinants of their photodynamic activity. The hexynyl trisulfonated zinc porphyrazine derivative (ZnNPcS(3)C(6)) showed the strongest in vitro photodynamic activity and therefore was further studied in an EMT-6 mouse tumour model. An i.v. dose of 1 micromol of ZnNPS(3)C(6) per kg, followed 24 h later by activation with light, induced 100% tumour necrosis within 24 h post-PDT. This treatment delayed tumour volume doubling time from 5 days to2 weeks, and gave 41% tumour cure3 weeks post-PDT. Applying the same light dose fractionated (5 min on, 2 min off), further improved tumour response, leading to a doubling time of 26 days and a 73% tumour cure. At the administered 1 micromol kg(-1) dye dose, no skin phototoxicity was observed and90% blood clearance was observed within 5 h post-injection. Compared to the analogous trisulfo monohexynyl zinc phthalocyanine, the new trisulfobenzo(mononaphthohexynyl)porphyrazine provided a broader range of excitation wavelengths, and improved photodynamic potency, while apparently being free of unwanted systemic side effects.