Search for new biologically active compounds: in vitro studies of antitumor and antimicrobial activity of dirhodium(II,II) paddlewheel complexes.

Four neutral Rh1-Rh4 complexes of the general formula [Rh2(CH3COO)4L2], where L is an N-alkylimidazole ligand, were synthesized and characterized using various spectroscopic techniques, and in the case of Rh4 the crystal structure was confirmed. Investigation of the interactions of these complexes with HSA by fluorescence spectroscopy revealed that the binding constants Kb are moderately strong (∼104 M-1), and site-marker competition experiments showed that the complexes bind to Heme site III (subdomain IB). Competitive binding studies for CT DNA using EB and HOE showed that the complexes bind to the minor groove, which was also confirmed by viscosity experiments. Molecular docking confirmed the experimental data for HSA and CT DNA. Antimicrobial tests showed that the Rh2-Rh4 complexes exerted a strong inhibitory effect on G+ bacteria B. cereus and G- bacteria V. parahaemolyticus as well as on the yeast C. tropicalis, which showed a higher sensitivity compared to fluconazole. The cytotoxic activity of Rh1-Rh4 complexes tested on three cancer cell lines (HeLa, HCT116 and MDA-MB-231) and on healthy MRC-5 cells showed that all investigated complexes elicited more efficient cytotoxicity on all tested tumor cells than on control cells. Investigation of the mechanism of action revealed that the Rh1-Rh4 complexes inhibit cell proliferation via different mechanisms of action, namely apoptosis (increase in expression of the pro-apoptotic Bax protein and caspase-3 protein in HeLa and HCT116 cells; changes in mitochondrial potential and mitochondrial damage; release of cytochrome c from the mitochondria; cell cycle arrest in G2/M phase in both HeLa and HCT116 cells together with a decrease in the expression of cyclin A and cyclin B) and autophagy (reduction in the expression of the protein p62 in HeLa and HCT116 cells).

Synthesis, characterization, DNA interactions and biological activity of new palladium(II) complexes with some derivatives of 2-aminothiazoles.

Newly palladium(II) complexes (C1, C2) with derivatives of 2-aminothiazoles (L1 = 2-amino-6-methylbenzothiazole, L2 = 2-amino-6-chlorobenzothiazole), general formula [PdL2Cl2] were synthesized and characterized by elemental microanalyses, IR, NMR spectroscopy and X-ray spectroscopy in case of [Pd(L2)2Cl2]. The kinetic of the substitution reactions of complexes and the nucleophiles, such as guanosine-5'-monophosphate (5'-GMP), tripeptide glutathione (GSH) and amino acid L-methionine (L-Met), were studied by stopped-flow technique. The complex C2 was always more reactive, while the order of the reactivity of the nucleophiles, due to the associative mode of the reaction, was L-Met > GSH > 5'-GMP. In order to determine the type of interactions between palladium(II) complexes and calf thymus DNA (CT-DNA), we used electronic absorption spectroscopy, viscosity measurements, and fluorescence spectroscopic studies, while interactions with bovine serum albumin (BSA) were determined only with fluorescence spectroscopic studies. The observed results confirmed that both complexes bound to DNA by groove binding. The significantly strong interaction with BSA, especially for complex C2, was also observed. In vitro cytotoxic activity was evaluated against four tumor cell lines, 4 T1, CT26, MDA-MB-468, HCT116 and mesenchymal stem cells (mMSC). C1 complex showed higher cytotoxic activity against CT26 cell line. Flow cytometry analysis showed that C1 stimulated apoptosis of tumor cells via inhibition of expression of antiapoptotic Bcl-2 molecule and decelerated proliferation by decreasing Cyclin-D and increasing expression of P21. In vitro antimicrobial activity for ligands and corresponding palladium(II) complexes was investigated by microdilution method and minimum inhibitory concentration (MIC) and minimum microbicidal concentration (MMC) were determined. Tested compounds exhibited selective and moderate activity.

Association between length of proestrus, follicular size, estrus behavior, and pregnancy rate in beef heifers subjected to fixed-time artificial insemination.

This study evaluated the relationship between proestrus length and follicular size, estrous behavior, and pregnancy rate in Bos taurus beef heifers subjected to fixed-time artificial insemination (FTAI). A total of 911 heifers received a synchronization treatment protocol for FTAI (J-Synch) consisting of an intravaginal progesterone device for 6 d, estradiol benzoate at the time of device insertion cloprostenol sodium and eCG at device removal and GnRH at the time of FTAI. The presence or absence of a corpus luteum (CL) was determined by ultrasonography at device insertion and all heifers were tail painted at device removal for estrus detection at the time of FTAI. For the establishment of different periods of proestrus length (i.e., interval from device removal to FTAI), GnRH was administered i.m. at 48 h (n = 308), 60 h (n = 290) or 72 h (n = 313) after device removal. The diameter of the largest follicle at the time of GnRH administration was determined by ultrasonography, expression of estrous was determined by percentage of tail paint removal, and FTAI was performed at the time of GnRH administration in all heifers. The diameter of the largest follicle was greater when GnRH/FTAI was performed at 72 or 60 h (12.9 ± 0.2 mm and 12.8 ± 0.1 mm, respectively) than at 48 h (12.2 ± 0.1 mm, P < 0.05). The proportion of heifers in estrus tended to be greater when GnRH/FTAI was performed at 72 h (77.0%, 137/178) than at 48 h (68.2%, 122/179; P = 0.06), and intermediate at 60 h (71.4%, 120/168). Pregnancy rate tended to be greater in heifers with the longest (72 h: 70.0%, 219/313) than the shortest (48 h: 63.6%, 196/308; P < 0.1) proestrus length, while 60 h proestrus length was intermediate (63.1%, 183/290; P= NS). Pregnancy rate was affected by the presence of a CL at device insertion (71.3%, 352/494 in heifers with a CL, vs. 59.0%, 246/417 for those without a CL; P < 0.01). For those heifers bearing a CL, pregnancy rate was greater in heifers with a 72 h proestrus length (77.0%, 134/174) than with 48 or 60 h proestrus length (67.7%, 107/158 and 68.5%, 111/162; respectively; P < 0.05). In heifers without a CL, proestrus length did not affect pregnancy rate. In summary, extending proestrus length by delaying the interval from device removal to GnRH/FTAI from 48 to 72 h, was associated with a greater diameter of the preovulatory follicle, greater proportion of heifers expressing estrus at the time of FTAI, and greater pregnancy rate in cycling beef heifers.

Influence of a Static Magnetic Field on the ⟨100⟩ Growth Rates of Sodium Chlorate Crystals from Aqueous Solution.

The results of the study of the influence of a static magnetic field of 55 ± 3 mT on the growth rates of diamagnetic sodium chlorate crystals in the direction ⟨100⟩ will be presented. Two groups of experiments were performed in the same solution supersaturation range of 0.89-1.78%, the first in zero field conditions, and the second in an applied magnetic field. The results show that crystals nucleated and grown in a static magnetic field have higher mean growth rates in the ⟨100⟩ direction than crystals in a zero field. Also, X-ray analyses suggest that crystals nucleated and grown in a magnetic field may have a higher lattice constant. Possible mechanisms and possible reasons for these phenomena are discussed.

Synthesis of new bile acid-fused tetrazoles using the Schmidt reaction.

A practical and high-yielding Schmidt reaction for the synthesis of fused tetrazoles from bile acid precursors was developed. Mild reaction conditions using TMSN3 instead of hydrazoic acid as an azide source produced good yields of the desired tetrazoles. These conditions could be applied to other steroidal precursors. Additionally, an improved methodology for the synthesis of different ketone and enone precursors from cholic acid, deoxycholic acid, and chenodeoxycholic acid was established. Newly obtained tetrazole derivatives were characterized by NMR and X-ray diffraction spectroscopy. In a number of cases, preliminary antiproliferative tests of new compounds showed strong and selective activity towards certain tumor cell lines.

Estradiol cypionate treatment in suckling/weaning and estrous cycling/anestrous beef cows subjected to fixed-time artificial insemination.

This study was conducted to evaluate the interaction between dose of estradiol cypionate (ECP) and ovarian status in beef cows on which different weaning/suckling regimens were imposed before fixed-time artificial insemination (FTAI). A total of 8070 estrous cycling and anestrous cows were subjected to three experiments, when calves were weaned early (Experiment 1), anti-suckling nose plates were applied for 9 or 10 days (Experiment 2), or there was continued suckling (Experiment 3). The cows were administered an estradiol/progesterone-based treatment regimen for FTAI and were treated with 0.5 or 1.0 mg of ECP im at the time of progesterone intravaginal device removal. Artificial insemination was performed from 46 to 56 h after the time of ECP treatment. Pregnancy per artificial insemination (P/AI) was affected by dose of ECP differentially in early-weaned and suckled cows. Whereas P/AI percentage was greater in early-weaned cows treated with 0.5 than 1.0 mg ECP (P < 0.05), P/AI percentage was greater for suckled cows treated with 1.0 than 0.5 mg ECP (P < 0.05). Although there were greater P/AI percentages in estrous cycling than anestrous cows (P < 0.05) when there was nose plate weaning and continuation of suckling, there was no difference between estrous cycling and anestrous cows (P = NS) when there was early weaning. Overall results indicate ECP administration affects fertility in a dose-dependent manner, suggesting an interaction between suckling and estrous cycling effects. As more critical the condition was (i.e., suckling anestrous cows), larger dose of estradiol was required.

Estradiol cypionate administered at the end of a progesterone-based protocol for FTAI induces ovulation and improves postovulatory luteal function and uterine environment in anestrous beef cows.

The objective of this study was to evaluate the effect of the administration of estradiol cypionate (ECP) at the end of an estradiol and progesterone-based protocol for fixed time artificial insemination (FTAI) on ovarian response and uterine function in postpartum anestrous beef cows. Multiparous suckled cows were randomly assigned to receive ECP at doses of 0 (control, n = 15), 0.5 (n = 15) or 1.0 mg (n = 15) im at the time of progesterone intravaginal insert removal. Serum 17ß-estradiol concentrations at 24 h after insert removal were greater (P < 0.05) in both ECP treatments than in controls. No differences in estradiol were found between 0.5 mg and control cows (P > 0.1) from 48 h after insert removal until ovulation, although greater (P < 0.05) concentrations were maintained until ovulation in 1.0 mg ECP treated cows. Maximum 17ß-estradiol concentration attained in each female was greater as ECP dose was greater (10.4 ± 0.4, 11.8 ± 0.5 and 13.5 ± 0.7, for control, 0.5 and 1.0 mg ECP treated cows, respectively; P < 005). Proportion of cows that ovulated tended to be greater (P = 0.06) in ECP treated than in control cows. Ovulation occurred earlier and the size of the ovulatory follicle was smaller (P < 0.05) for 1.0 mg but not for 0.5 mg (P > 0.1) when compared with control cows. After ovulation (Day 13 and 14), serum progesterone concentrations were greater (P < 0.05) in 0.5 and 1.0 mg ECP than control cows. Uterine environment on Day 6 after ovulation was affected by treatment; transcript expression of three of nine evaluated genes (i.e., estrogen, IGF-1 and insulin receptors genes) were upregulated (P < 0.05) after ECP treatment. In conclusion, ECP administration at progesterone insert removal in anestrous cows i) induces greater serum estradiol concentrations and tended to induce greater ovulation rate, ii) acts in a dose-dependent manner, as ECP dose increases ovulation occurs earlier and the size of the ovulatory follicle is smaller, iii) improves postovulatory luteal function and affects uterine gene expression. Altogether, this information contributes with the understanding of the effect of preovulatory estradiol exposure on ovulation and postovulatory ovarian and uterine function in anestrous beef cows.

Synthesis, characterization, HSA/DNA interactions and antitumor activity of new [Ru(η6-p-cymene)Cl2(L)] complexes.

Three new ruthenium(II) complexes were synthesized from different substituted isothiazole ligands 5-(methylamino)-3-pyrrolidine-1-ylisothiazole-4-carbonitrile (1), 5-(methylamino)-3-(4-methylpiperazine-1-yl)isothiazole-4-carbonitrile (2) and 5-(methylamino)-3-morpholine-4-ylisothiazole-4-carbonitrile (3): [Ru(η6-p-cymene)Cl2(L1)]·H2O (4), [Ru(η6-p-cymene)Cl2(L2)] (5) and [Ru(η6-p-cymene)Cl2(L3)] (6). All complexes were characterized by IR, UV-Vis, NMR spectroscopy, and elemental analysis. The molecular structures of all ligands and complexes 4 and 6 were determined by an X-ray. The results of the interactions of CT-DNA (calf thymus deoxyribonucleic acid) and HSA (human serum albumin) with ruthenium (II) complexes reveal that complex 4 binds well to CT-DNA and HSA. Kinetic and thermodynamic parameters for the reaction between complex and HSA confirmed the associative mode of interaction. The results of Quantum mechanics (QM) modelling and docking experiments toward DNA dodecamer and HSA support the strongest binding of the complex 4 to DNA major groove, as well as its binding to IIa domain of HSA with the lowest ΔG energy, which agrees with the solution studies. The modified GOLD docking results are indicative for Ru(p-cymene)LCl··(HSA··GLU292) binding and GOLD/MOPAC(QM) docking/modelling of DNA/Ligand (Ru(II)-N(7)dG7) covalent binding. The cytotoxic activity of compounds was evaluated by MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide) assay. Neither of the tested compounds shows activity against a healthy MRC-5 cell line while the MCF-7 cell line is the most sensitive to all. Compounds 3, 4 and 5 were about two times more active than cisplatin, while the antiproliferative activity of 6 was almost the same as with cisplatin. Flow cytometry analysis showed the apoptotic death of the cells with a cell cycle arrest in the subG1 phase.

A First Step to a Biomarker of Curative Surgery in Colorectal Cancer by Liquid Biopsy of Methylated Septin 9 Gene.

OBJECTIVES: To confirm that patients affected by colorectal cancer have the V2 region of Septin 9 (SEPT9) gene hypermethylated in the circulating free DNA from a peripheral blood sample before surgery and to determine if this hypermethylated DNA disappears from the patients after complete resection of the tumour. METHODS: Plasma from 10 patients with colorectal cancer was collected preoperative and three months after surgery. The analysis of the methylation status of the promoter region of the SEPT9 gene was performed using a 7500 Fast Real-Time PCR System. RESULTS: Hypermethylation of SEPT9 gene was detected in 8 out of 10 preoperative samples (one negative result was probed to be a Lynch syndrome) and in 4 out of 10 postoperative samples matching with the cases of recurrence or persistence of disease. This means that, in this sample, the preoperative sensitivity and specificity of the test were 88.9% and 100%, respectively, and there is 100% correlation between the positive results of the SEPT9 test and a recurrence/persistence of the disease in patients after surgical resection. CONCLUSIONS: Our study shows that circulating hypermethylated SEPT9 is a specific colorectal cancer biomarker. This hypermethylated SEPT9 DNA disappears around three months after surgery and that circulating hypermethylated SEPT9 may be the first noninvasive marker for postsurgical diagnosis; this conclusion must be confirmed with a more significant number of patients.

Effect of equine chorionic gonadotropin (eCG) administration and proestrus length on ovarian response, uterine functionality and pregnancy rate in beef heifers inseminated at a fixed-time.

The objective of the present study was to evaluate the effect of equine chorionic gonadotropin (eCG) administration associated to different proestrus lengths for Fixed-time AI (FTAI) in beef heifers. In Experiment 1, pre-pubertal heifers (n = 46) received a 6-day estradiol/progesterone-based treatment (J-Synch protocol), and were then allocated into four experimental groups in a 2 × 2 factorial design, to receive or not receive eCG (300 IU) at the time of intravaginal progesterone device removal, and to receive GnRH at 48 h or 72 h after device removal (to induce shortened and prolonged proestrus length, respectively). Endometrial samples were obtained 6 d after ovulation from the cranial portion of the uterine horn. The eCG administration induced greater serum estradiol-17ß concentrations before ovulation (P < 0.05) and greater proportion of heifers bearing a competent corpus luteum after ovulation (P = 0.054). Delaying GnRH administration from 48 h to 72 h induced a longer interval from device removal to ovulation (i.e., prolonged proestrus; P < 0.05), larger diameter of the ovulatory follicle, and greater progesterone concentrations on Day 10-11 after ovulation. Heifers in eCG + GnRH72h group had more uterine receptors in luminal epithelium than those in eCG + GnRH48h group (PR and ERα), and than those in No eCG + GnRH72h group (PR) (P < 0.05). No effect of eCG or GnRH treatments was found in endometrial gene expression of progesterone and estrogen receptors. In Experiment 2, a total of 2,598 heifers received the J-Synch protocol associated or not with eCG administration at device removal, followed by FTAI/GnRH at 60 or 72 h after device removal (i.e., prolonged proestrus protocol). Heifers that received eCG had greater P/AI than those not receiving eCG (P < 0.05) and there was an interaction between eCG treatment and time of FTAI. The lowest P/AI was found in those heifers that received FTAI/GnRH at 72 h without eCG treatment at device removal (P < 0.05), and no differences were found between the other experimental groups. In conclusion, prolonging the length of proestrus in J-Synch protocol improves ovulatory follicular diameter and luteal function; and the administration of eCG at device removal improves preovulatory estradiol concentrations and luteal function. Finally, P/AI was enhanced by eCG treatment and the improvement was more evident when FTAI/GnRH was performed at 72 h after device removal.

Local influence of the corpus luteum on the ipsilateral oviduct and early embryo development in the ewe.

The objective of this study was to evaluate the local effect of the corpus luteum (CL) on ipsilateral oviduct-uterus functionality and early embryo development in ewes. A total of 499 embryos were transferred on Day 1 after in vitro fertilization into the ipsilateral (n = 250) and contralateral oviducts (n = 249) of 13 ewes on Day 1 after ovulation (18-20 embryos per oviduct). On Day 6, their reproductive tracts were collected and their uterine horns were flushed for embryo recovery. More recovered embryos, a higher proportion of blastocysts, and more viable embryos were collected when the embryos were transferred into the ipsilateral oviducts (P < 0.05). In addition, almost five times higher P4 concentrations and significantly lower E2 concentrations, with higher P4:E2 ratio, were found in the ipsilateral than contralateral oviductal tissue (P < 0.05). Furthermore, a higher concentration of adiponectin was found in the ipsilateral uterine tissue macerates than in the contralateral side to the CL. The ipsilateral oviductal tissue had a lower expression of PGR and IGFBP5, but the transcript expression of ADIPOR1 was higher in the ipsilateral oviductal tissue. In the uterus, the mRNA expression of ESR1, IGFBP3, IGFBP5, and LEPR was higher or tended to be higher in the ipsilateral than contralateral uterine tissue. Uterine flushing fluid collected from the ipsilateral uterine horn had lower insulin concentrations than the contralateral horn, while no differences were found in the P4 and E2 concentrations. In conclusion, on Day 6 post-ovulation, P4 was elevated in the ipsilateral oviductal tissue, embryo development was advanced, and differential gene expression of PGR, ESR1, IGFBP3, IGFBP5, LEPR, and ADIPOR1 in the oviductal or uterine tissue was found between the ipsilateral and contralateral side. This study demonstrates local regulation of the ovary on the ipsilateral oviduct/uterine horn in the ewe.

Synthesis of Camphor-Derived Bis(pyrazolylpyridine) Rhodium(III) Complexes: Structure-Reactivity Relationships and Biological Activity.

Two novel rhodium(III) complexes, namely, [RhIII(X)Cl3] (X = 2 2,6-bis((4 S,7 R)-7,8,8-trimethyl-4,5,6,7-tetrahydro-1 H-4,7-methanoindazol-3-yl)pyridine or 2,6-bis((4 S,7 R)-1,7,8,8-tetramethyl-4,5,6,7-tetrahydro-1 H-4,7-methanoindazol-3-yl)pyridine), were synthesized from camphor derivatives of a bis(pyrazolylpyridine), tridentate nitrogen-donor chelate system, giving [RhIII(H2L*)Cl3] (1a) and [RhIII(Me2L*)Cl3] (1b). A rhodium(III) terpyridine (terpy) ligand complex, [RhIII(terpy)Cl3] (1c), was also synthesized. By single-crystal X-ray analysis, 1b crystallizes in an orthorhombic P212121 system, with two molecules in the asymmetric unit. Tridentate coordination by the N,N,N-donor localizes the central nitrogen atom close to the rhodium(III) center. Compounds 1a and 1b were reactive toward l-methionine (l-Met), guanosine-5'-monophosphate (5'-GMP), and glutathione (GSH), with an order of reactivity of 5'-GMP > GSH > l-Met. The order of reactivity of the RhIII complexes was: 1b> 1a > 1c. The RhIII complexes showed affinity for calf thymus DNA and bovine serum albumin by UV-vis and emission spectral studies. Furthermore, 1b showed significant in vitro cytotoxicity against human epithelial colorectal carcinoma cells. Since the RhIII complexes have similar coordination modes, stability differences were evaluated by density functional theory (DFT) calculations (B3LYP(CPCM)/LANL2DZp). With (H2L*) and (terpy) as model ligands, DFT calculations suggest that both tridentate ligand systems have similar stability. In addition, molecular docking suggests that all test compounds have affinity for the minor groove of DNA, while 1b and 1c have potential for DNA intercalation.

Evaluation of A-ring fused pyridine d-modified androstane derivatives for antiproliferative and aldo-keto reductase 1C3 inhibitory activity.

New A-ring pyridine fused androstanes in 17a-homo-17-oxa (d-homo lactone), 17α-picolyl or 17(E)-picolinylidene series were synthesized and validated by X-ray crystallography, HRMS, IR and NMR spectroscopy. Novel compounds 3, 5, 8 and 12 were prepared by treatment of 4-en-3-one or 4-ene-3,6-dione d-modified androstane derivatives with propargylamine catalyzed by Cu(ii), and evaluated for potential anticancer activity in vitro using human cancer cell lines and recombinant targets of steroidal anti-cancer drugs. Pyridine fusion to position 3,4 of the A-ring may dramatically enhance affinity of 17α-picolyl compounds for CYP17 while conferring selective antiproliferative activity against PC-3 cells. Similarly, pyridine fusion to the A-ring of steroidal d-homo lactones led to identification of new inhibitors of aldo-keto reductase 1C3, an enzyme targeted in acute myeloid leukemia, breast and prostate cancers. One A-pyridine d-lactone steroid 5 also has selective submicromolar antiproliferative activity against HT-29 colon cancer cells. None of the new derivatives have affinity for estrogen or androgen receptors in a yeast screen, suggesting negligible estrogenicity and androgenicity. Combined, our results suggest that A-ring pyridine fusions have potential in modulating the anticancer activity of steroidal compounds.

Effects of extending the length of pro-oestrus in an oestradiol- and progesterone-based oestrus synchronisation program on ovarian function, uterine environment and pregnancy establishment in beef heifers.

The aim of the present study was to investigate the effects of a strategy for extending pro-oestrus (the interval between luteolysis and ovulation) in an oestrus synchronisation protocol (named J-Synch) in beef heifers on follicular growth, sexual steroid concentrations, the oestrogen receptor ERα and progesterone receptors (PR) in the uterus, insulin-like growth factor (IGF) 1 and pregnancy rates. In Experiment 1, heifers treated with the new J-Synch protocol had a longer pro-oestrus period than those treated with the conventional protocol (mean (±s.e.m.) 93.7±12.9 vs 65.0±13.7h respectively; P<0.05). The rate of dominant follicle growth from the time of progesterone device removal to ovulation was greater in heifers in the J-Synch than conventional group (P<0.05). Luteal area and serum progesterone concentrations were greater in the J-Synch Group (P<0.05) for the 12 days after ovulation. Progesterone receptor (PGR) staining on Day 6 after ovulation in the uterine stroma was lower in the J-Synch than conventional group (P<0.05), and the expression of PR gene (PGR) and IGF1 gene tended to be lower in J-Synch-treated heifers (P<0.1). In Experiment 2 (n=2349), the pregnancy rate 30-35 days after fixed-time AI (FTAI) was greater for heifers in the J-Synch than conventional group (56.1% vs 50.7% respectively). In conclusion, our strategy for extending pro-oestrus (i.e. the J-Synch protocol) significantly improves pregnancy establishment in beef heifers. This improvement was related to an increased rate of growth of the dominant ovulatory follicle, greater progesterone concentrations during the ensuing luteal phase and different uterine patterns of PGR and IGF1, which may have favoured embryo development and pregnancy establishment.

Bone marrow mesenchymal stem cells as nuclear donors improve viability and health of cloned horses.

INTRODUCTION: Cell plasticity is crucial in cloning to allow an efficient nuclear reprogramming and healthy offspring. Hence, cells with high plasticity, such as multipotent mesenchymal stem cells (MSCs), may be a promising alternative for horse cloning. In this study, we evaluated the use of bone marrow-MSCs (BM-MSCs) as nuclear donors in horse cloning, and we compared the in vitro and in vivo embryo development with respect to fibroblasts. MATERIALS AND METHODS: Zona-free nuclear transfer was performed using BM-MSCs (MSC group, n=3432) or adult fibroblasts (AF group, n=4527). Embryos produced by artificial insemination (AI) recovered by uterine flushing and transferred to recipient mares were used as controls (AI group). RESULTS: Blastocyst development was higher in the MSC group than in the AF group (18.1% vs 10.9%, respectively; p<0.05). However, pregnancy rates and delivery rates were similar in both cloning groups, although they were lower than in the AI group (pregnancy rates: 17.7% [41/232] for MSC, 12.5% [37/297] for AF and 80.7% [71/88] for AI; delivery rates: 56.8% [21/37], 41.5% [17/41] and 90.1% [64/71], respectively). Remarkably, the gestation length of the AF group was significantly longer than the control (361.7±10.9 vs 333.9±8.7 days), in contrast to the MSC group (340.6±8.89 days). Of the total deliveries, 95.2% (20/21) of the MSC-foals were viable, compared to 52.9% (9/17) of the AF-foals (p<0.05). In addition, the AF-foals had more physiological abnormalities at birth than the MSC-foals; 90.5% (19/21) of the MSC-delivered foals were completely normal and healthy, compared to 35.3% (6/17) in the AF group. The abnormalities included flexural or angular limb deformities, umbilical cord enlargement, placental alterations and signs of syndrome of neonatal maladjustment, which were treated in most cases. CONCLUSION: In summary, we obtained 29 viable cloned foals and found that MSCs are suitable donor cells in horse cloning. Even more, these cells could be more efficiently reprogrammed compared to fibroblasts.

Equine chorionic gonadotropin administration after insemination affects luteal function and pregnancy establishment in postpartum anestrous beef cows.

Two experiments were conducted with the aim of determining the effect of equine chorionic gonadotropin (eCG) administration on day 14 after insemination on ovarian response and pregnancy establishment in postpartum anestrous beef cows. In both experiments, cows were subjected to a progesterone- and estradiol-based treatment for fixed-time artificial insemination (FTAI) and were randomly allocated into 4 groups to receive or not receive eCG (400 IU) at the time of device removal and/or at 14 d after FTAI. In experiment 1, from day 14 to 22, daily ultrasonographic determinations were performed to monitor ovarian dynamics, and blood was collected to determine hormone concentrations in 60 cows. In experiment 2, confirmation of pregnancy was performed at 30 and 60 d after FTAI in 1,060 anestrous cows assigned to the same experimental design. Cows that received eCG on day 14 after FTAI showed increases in corpus luteum area (P < 0.01), follicle diameter (P < 0.05), serum progesterone concentrations (P < 0.01), and estradiol-17ß concentrations (P < 0.01), compared with cows that did not receive eCG on day 14. Pregnancy rate on day 30 was greater in those cows that received both eCG treatments (ie, at device removal and 14 d after insemination) than in those that did not receive eCG treatment (P < 0.05). In conclusion, eCG administered on day 14 after FTAI increases serum progesterone concentrations during the critical period of pregnancy in anestrous cows, and this second eCG treatment seems to have a positive effect on achieving pregnancy.

Dynamic simulation and Doppler Ultrasonography validation of blood flow behavior in Abdominal Aortic Aneurysm.

Criteria for rupture prediction of Abdominal Aortic Aneurysm (AAA) are based only on the diameter of AAA. This method does not consider complex hemodynamic forces exerted on AAA wall. The methodology used in our study combines Computer-Aided Design (CAD) with Computational Fluid Dynamics (CFD). Three-dimensional vascular structures reconstructions were based on Computed Tomography (CT) images and CAD. CFD theory was used for mathematical modeling and simulations. In this way, dynamic behavior of blood flow in bounded three-dimensional space was described. Doppler Ultrasonography (US) was used for model results validation. All simulations were based on medical investigation of 4 patients (male older than 65years) with diagnosed AAA. Good correspondence between computed velocities in AAA and measured values with Doppler US (Patient 1 0.60m·s-1 versus 0.61m·s-1, Patient 2 0.80m·s-1 versus 0.80m·s-1, Patient 3 0.75m·s-1 versus 0.78m·s-1, Patient 4 0.50m·s-1 versus 0.49m·s-1) was noticed. The good agreement between measured and simulated velocities validates our methodology and the other data available from simulations (eg. von Misses stress) could be used to provide useful information about the possibility of AAA rupture.

Kinetic and mechanistic study on the reactions of ruthenium(ii) chlorophenyl terpyridine complexes with nucleobases, oligonucleotides and DNA.

In this study, we investigated the ability of Ru(ii) polypyridyl complexes to act as DNA binders. The substitution reactions of three Ru(ii) chlorophenyl terpyridine complexes, i.e. [Ru(Cl-Ph-tpy)(en)Cl]Cl (1), [Ru(Cl-Ph-tpy)(dach)Cl]Cl (2) and [Ru(Cl-Ph-tpy)(bpy)Cl]Cl (3) (Cl-Ph-tpy = 4'-(4-chlorophenyl)-2,2':6',2''-terpyridine, en = 1,2-diaminoethane, dach = 1,2-diaminocyclohexane, bpy = 2,2'-bipyridine), with a mononucleotide guanosine-5'-monophosphate (5'-GMP) and oligonucleotides such as fully complementary 15-mer and 22-mer duplexes with a centrally located GG-binding site for DNA, and fully complementary 13-mer duplexes with a centrally located GG-binding site for RNA were studied quantitatively by UV-Vis spectroscopy. Duplex RNA reacts faster with complexes 1-3 than duplex DNA, while shorter duplex DNA (15mer GG) reacts faster compared with 22mer GG duplex DNA. The measured enthalpies and entropies of activation (ΔH≠ > 0, ΔS≠ < 0) support an associative mechanism for the substitution process. 1H NMR spectroscopy studies performed on complex 3 demonstrated that after the hydrolysis of the Cl ligand, it is capable to interact with guanine derivatives (i.e., 9-methylguanine (9MeG) and 5'-GMP) through N7, forming monofunctional adducts. The molecular structure of the cationic compound [Ru(Cl-Ph-tpy)(bpy)Cl]Cl (3) was determined in the solid state by X-ray crystallography. The interactions of 1-3 with calf thymus (CT) and herring testes (HT) DNA were examined by stopped-flow spectroscopy, in which HT DNA was sensibly more reactive than CT DNA. The reactivity towards the formation of Ru-DNA adducts was also revealed by a gel mobility shift assay, showing that complexes 1 and 2 have a stronger DNA unwinding ability compared to complex 3. Overall, the complexes with bidentate aliphatic diamines proved to be superior to those with bpy in terms of capability to bind to the here studied biomolecules.

Synthesis, structural analysis and antitumor activity of novel 17α-picolyl and 17(E)-picolinylidene A-modified androstane derivatives.

The heterocyclic ring at C-17 position of the androstane compounds plays an important role in biological activity. The aim of the present study was to synthesize and evaluate potential antitumor activity of different A-modified 17α-picolyl and 17(E)-picolinylidene androstane derivatives. In several synthetic steps, novel derivatives bearing the hydroximino, nitrile or lactame functions in A-ring were synthesized and characterized according to the spectral data, by mass analysis as well as XRD analysis (compounds 6, 13 and 15). The structurally most promising compounds 6, 11-17 were investigated as antitumor agents. The in vitro antiproliferative activity was evaluated against six human cancer cell lines: estrogen receptor negative (ER-) breast adenocarcinoma (MDA-MB-231); estrogen receptor positive (ER+) breast adenocarcinoma (MCF-7); prostate cancer (PC-3); human cervical carcinoma (HeLa); lung adenocarcinoma (A549) and colon adenocarcinoma (HT-29) using MTT assay. The results of the 48h incubation time in vitro tests showed that compound 15 was the most effective against PC-3 (IC50 6.6µM), compound 17 against MCF-7 (IC50 7.9µM) cells, while compound 16 exhibited strong antiproliferative effect against both, MCF-7 (IC50 1.7µM) and PC-3 (IC50 8.7µM) cancer cells. It was also found that compounds 16 and 17 induced apoptosis in MCF-7 cells (dicyano derivative 17 stronger then dioxime 16 and reference formestane), with no distinct changes in the cell cycle of MCF-7 cells.