Determination of mildronate by LC–MS/MS and its application to a pharmacokinetic study in healthy Chinese volunteers

A simple, rapid and accurate liquid chromatography–tandem mass spectrometry (LC–MS/MS) method has been developed and validated for the determination of mildronate in human plasma. Following a simple protein precipitation with methanol, the analyte was separated on a C₁₈ column by isocratic elution with methanol and 10 mM ammonium acetate (55:45; v/v), and then analyzed by mass spectrometry in the positive ion MRM mode. Good linearity was achieved over a wide range of 0.01–20 μg/mL. The intra- and inter-batch precisions (as RSD, %) were less than 7.1%. The average extraction recovery was 87.5%. The method described above has been used, for the first time, to reveal the pharmacokinetics of mildronate injection in healthy subjects. After single intravenously administration of 250, 500 and 1000 mg mildronate, the elimination half-life (t_1/2) were (5.56 ± 1.55), (6.46 ± 1.07) and (6.55 ± 1.17) h, respectively. The Student–Newman–Keuls test results showed that peak plasma concentration (C_max) and the area under the plasma concentration versus time curve from time 0 to 24 h (AUC_0–24) were both linearly related to dose. The pharmacokinetics of mildronate fitted the linear dynamic feature over the dose range studied. The essential pharmacokinetic parameters of multidoses administration intravenously (500 mg, b.i.d) were as follows: t_1/2 was (15.34 ± 3.14) h; C_max was (25.50 ± 3.63) μg/mL; AUC_0–24 was (58.56 ± 5.57) mg h/L. The t_1/2 and AUC of multidoses administration intravenously were different from those of single-dose administration significantly. These findings suggested that accumulation of mildronate in plasma occurred.     

Mechanical properties of the patellar tendon in adults and children

It is not currently known how the mechanical properties of human tendons change with maturation in the two sexes. To address this, the stiffness and Young's modulus of the patellar tendon were measured in men, women, boys and girls (each group, n=10). Patellar tendon force (F_pt) was calculated from the measured joint moment during a ramped voluntary isometric knee extension contraction, the antagonist knee extensor muscle co-activation quantified from its electromyographical activity, and the patellar tendon moment arm measured from magnetic resonance images. Tendon elongation was imaged using the sagittal-plane ultrasound scans throughout the contraction. Tendon cross-sectional area was measured at rest from ultrasound scans in the transverse plane. Maximal F_pt and tendon elongation were (mean±SE) 5453±307 N and 5±0.5 mm for men, 3877±307 N and 4.9±0.6 mm for women, 2017±170 N and 6.2±0.5 mm for boys and 2169±182 N and 5.9±0.7 mm for girls. In all groups, tendon stiffness and Young's modulus were examined at the level that corresponded to the maximal 30% of the weakest participant's F_pt and stress, respectively; these were 925–1321 N and 11.5–16.5 MPa, respectively. Stiffness was 94% greater in men than boys and 84% greater in women than girls (p<0.01), with no differences between men and women, or boys and girls (men 1076±87 N/mm; women 1030±139 N/mm; boys 555±71 N/mm and girls 561.5±57.4 N/mm). Young's modulus was 99% greater in men than boys (p<0.01), and 66% greater in women than girls (p<0.05). There were no differences in modulus between men and women, or boys and girls (men 597±49 MPa; women 549±70 MPa; boys 255±42 MPa and girls 302±33 MPa). These findings indicate that the mechanical stiffness of tendon increases with maturation due to an increased Young's modulus and, in females due to a greater increase in tendon cross-sectional area than tendon length.     

Influence of verapamil on pharmacokinetics and transplacental transfer of ivermectin in sheep

In pregnant sheep at 120–128 days of gestational age, a study was done to evaluate the effect of the co-administration of verapamil (Vpm) and ivermectin (IVM) on the maternal and fetal disposition kinetics after intravenous administration of IVM. Ten pregnant Suffolk Down sheep of 63.5 ± 6.6 kg body weight (bw) were surgically prepared to insert polyvinyl catheters in the fetal femoral artery and vein and amniotic sac. The ewes were randomly assigned to two experimental groups. In Group 1 (control), 5 ewes were treated with an intravenous bolus of 0.2 mg IVM/kg bw. In Group 2 (Vpm-IVM), 5 ewes were subcutaneously treated with Vpm (2.5 mg/kg × 3 doses at 12 h intervals) and 0.2 mg/kg IVM by intravenous route. Maternal and fetal blood samples were taken before and after IVM administration during a 144 h post-treatment period. Samples were analyzed by liquid chromatography (HPLC). A non-compartmental pharmacokinetic analysis was performed and statistical differences were determined using the Mann–Whitney U-test.Significantly higher IVM levels in maternal plasma concentrations were determined after co-administration of Vpm/IVM compared with the group treated with IVM alone. Significant decreases in the volume of distribution and in the half-life of elimination (t_½β) were observed in the Vpm/IVM treated group. A significantly faster (T_max) and higher (C_max) (P < 0.05) increase in IVM fetal plasma concentrations were observed in the group of fetuses from pregnant ewes treated with Vpm/IVM. The results of our study support the hypothesis that pharmacological blockage of P-glycoprotein with Vpm can increase the transfer of IVM to fetal circulation.     

The effect of time left alone at home on dog welfare

The aim of this study was to investigate the effect of time left alone on dog behaviour and cardiac activity. Twelve privately owned dogs, with no history of separation related behaviour problems, were video-recorded on three different occasions when left alone in their home environment. The treatments lasted for 0.5 h (T_0.5); 2 h (T₂) and 4 h (T₄). Video-recording started 10 min before the owner left the house and continued until 10 min after the owner returned, so that interactions between dog and owner as well as behaviour during separation could be studied. Data on heart rate (HR) and heart rate variability (HRV) were collected within the same time period in each treatment. In addition to analysing behaviours separately, behaviours were also grouped together and defined as new variables; physically active, attentive behaviour, vocal, interaction initiated by owner and interaction initiated by dog. There were no differences in behaviour between treatments at equivalent time intervals until the owner returned, although a number of differences were observed at reunion with the owner. Dogs showed a higher frequency of physical activity (P < 0.05) and attentive behaviour (P < 0.01) in T₂ (0.37 ± 0.07; 0.52 ± 0.08, mean frequency of occurrence/15 s ± SE) and T₄ (0.48 ± 0.08; 0.48 ± 0.07) compared to T_0.5 (0.20 ± 0.07; 0.21 ± 0.05). They also showed more tail wagging (P < 0.01) and interacted more with their owners (P < 0.01) in T₂ (0.27 ± 0.08; 0.47 ± 0.09) and T₄ (0.26 ± 0.04; 0.42 ± 0.09) compared to T_0.5 (0.09 ± 0.04; 0.14 ± 0.03). After a longer time of separation, the dogs also showed higher frequencies of lip licking (P < 0.05) and body shaking (P < 0.05) at the owner's return (T_0.5 = 0.09 ± 0.05; T₂ = 0.24 ± 0.08; T₄ = 0.27 ± 0.06 and T_0.5 = 0.03 ± 0.01; T₂ = 0.08 ± 0.03; T₄ = 0.07 ± 0.01, respectively). There was a tendency for higher HR (P < 0.1) during the first and second minute after reunion in T₂ (127.6 ± 1.25, mean bpm ± SE; 111.3 ± 1.24) compared to T_0.5 (106.2 ± 1.06; 87.5 ± 1.02). According to the results of this study, the effect of time left alone was shown by a more intense greeting behaviour by the dog towards their owner as well as by a higher frequency of physical activity and attentive behaviour when the owner returned, already after 2 h of separation. Although this study cannot distinguish between whether dogs were aware of the length of time they were alone (but did not signal it) or whether they were unaware until reminded of it by the return of their owner, it does confirm that dogs are affected by the duration of time at home alone.     

Time-lapse videography of human oocytes following intracytoplasmic sperm injection: Events up to the first cleavage division

A total of 341 fertilized and 37 unfertilized oocytes from 63 intracytoplasmic sperm injection (ICSI) treatment cycles were included for retrospective assessment using the Embryoscope™ time-lapse video system. The second polar body (pb2) extrusion occurred at 2.9 ± 0.1 h (range 0.70–10.15 h) relative to sperm injection. All oocytes reduced in size following sperm injection (p < 0.05) with shrinkage ceasing after 2 h in the unfertilized and at pb2 extrusion in the fertilized oocytes. Pb2 extrusion was significantly delayed for women aged >38 years compared to those <35 years (3.4 ± 0.2 vs. 2.8 ± 0.1, p < 0.01) or 35–38 years (3.4 ± 0.2 vs. 2.8 ± 0.1, p < 0.01), but timing was not related to the Day 3 morphological grades (1–4) of subsequent embryos (2.9 ± 0.1, 2.9 ± 0.1, 2.8 ± 0.2 and 3.0 ± 0.1; p > 0.05 respectively). A shorter time of first cleavage division relative to either sperm injection or pb2 extrusion is associated with both top grade (AUC = 0.596 or 0.601, p = 0.006 or 0.004) and usable embryos (AUC = 0.638 or 0.632, p = 0.000 respectively) on Day 3. In summary, (i) pb2 of human oocytes extrudes at various times following sperm injection, (ii) the timing of pb2 extrusion is significantly delayed when female age >38 years, but not related to subsequent embryo development, (iii) all human oocytes reduce in size following sperm injection, (iv) completion of pb2 extrusion in the fertilized oocytes is a pivotal event in terminating shrinkage of the vitellus, and (v) time to first cleavage division either from sperm injection or pb2 extrusion is a significant predictive marker for embryo quality on Day 3.     

Influences of short-term pre-slaughter dietary manipulation in sheep and goats on pH and microbial loads of gastrointestinal tract

Sheep (BW = 39.9 kg, n = 16) and goats (BW = 32.8 kg, n = 16) were used in a completely randomized design to determine the effect of short-term pre-slaughter diet and feed deprivation (FD) time on pH and microbial loads in the gastrointestinal tract (GIT) contents. In a 2 × 2 × 2 factorial treatment arrangement, the main effects of species, diet, and FD time prior to slaughter and their interactions were studied. Animals were fed either a hay or concentrate diet for 4 d and then feed deprived for either 12 or 24-h prior to slaughter. The pH of rumen and colon contents as well as weight of GIT was measured. The contents of rumen and rectum were also sampled for microbial analysis. The GIT of sheep (1.82 kg) was heavier (P < 0.05) than that of goats (1.46 kg). The 12-h FD group (1.74 kg) had a higher (P < 0.05) GIT weight than the 24-h FD group (1.53 kg). Hay-fed animals had higher (P < 0.05) rumen (7.08 vs. 6.43) and colon pH values (7.02 vs. 6.56) than those of the concentrate-fed animals. The 24-h FD group (3.39 ± 0.272 log₁₀CFU/g) contained more (P < 0.05) Escherichia coli in the rumen than did the 12-h FD (2.17 ± 0.272 log₁₀CFU/g) group. The concentrate-fed animals (3.49 ± 0.289 log₁₀CFU/g) had higher (P < 0.05) coliform counts in the rumen than the hay-fed animals (2.43 ± 0.289 log₁₀CFU/g). The 24-h FD group (3.42 ± 0.289 log₁₀CFU/g) had a higher (P < 0.05) concentration of coliform than did the 12-h FD group (2.50 ± 0.289 log₁₀CFU/g). The 24-h FD group (3.31 ± 0.259 log₁₀CFU/g) also had higher (P < 0.05) Enterobacteriaceae counts in the rumen than did in the 12-h FD group (2.47 ± 0.259 log₁₀CFU/g). Goats (5.71 ± 0.158 log₁₀CFU/g) had lower (P < 0.05) total plate counts in the rumen compared to sheep (6.27 ± 0.158 log₁₀CFU/g). The concentrate-fed animals had higher (P < 0.05) E. coli (6.44 vs. 4.01 ± 0.468 log₁₀CFU/g), total coliform (6.74 vs. 4.16 ± 0.469 log₁₀CFU/g), Enterobacteriaceae (6.93 vs. 3.83 ± 0.651 log₁₀CFU/g), and total plate counts (7.79 vs. 7.28 ± 0.170 log₁₀CFU/g) in the rectum than the hay-fed animals. The results indicate that microbial loads in the GIT of small ruminants may be reduced by either feeding hay for 4 d or depriving feed for 12-h prior to slaughter.     

Oleanolic acid (OA) as an antileishmanial agent: Biological evaluation and in silico mechanistic insights

Although a worldwide health problem, leishmaniasis is considered a highly neglected disease, lacking efficient and low toxic treatment. The efforts for new drug development are based on alternatives such as new uses for well-known drugs, in silico and synthetic studies and naturally derived compounds. Oleanolic acid (OA) is a pentacyclic triterpenoid widely distributed throughout the Plantae kingdom that displays several pharmacological activities. OA showed potent leishmancidal effects in different Leishmania species, both against promastigotes (IC_{50 L. braziliensis} 30.47 ± 6.35 μM; IC_{50 L. amazonensis} 40.46 ± 14.21 μM; IC_{50 L. infantum} 65.93 ± 15.12 μM) and amastigotes (IC_{50 L. braziliensis} 68.75 ± 16.55 μM; IC_{50 L. amazonensis} 38.45 ± 12.05 μM; IC_{50 L. infantum} 64.08 ± 23.52 μM), with low cytotoxicity against mouse peritoneal macrophages (CC₅₀ 235.80 ± 36.95 μM). Moreover, in silico studies performed to evaluate OA molecular properties and to elucidate the possible mechanism of action over the Leishmania enzyme sterol 14α-demethylase (CYP51) suggested that OA interacts efficiently with CYP51 and could inhibit the ergosterol synthesis pathway. Collectively, these data indicate that OA is a good candidate as leading compound for the development of a new leishmaniasis treatment.     

Influence of physiological concentrations of androgens on the developmental competence and sex ratio of in vitro produced bovine embryos

This study was designed to determine if the addition of androgens at ovarian follicular fluid (FF) concentrations to oocyte maturation media would alter the development and sex ratio of bovine embryos. To maximize hormone bioavailability, oil was removed and glass culture dishes were used during in vitro maturation (IVM) phase; this modified system was then used in the present experiment along with the standard IVM system utilizing plastic containers and incubation under oil. Ethanol (0.2%) was the vector for steroid hormone delivery. Oocytes were incubated for 22 h in the presence of two doses (“low” and “high”) of androstenedione (A₄) or testosterone (T); the doses were based on the concentrations of both androgens in preovulatory bovine follicles (A₄: 337.5 and 562.5 ng/ml; T: 22.2 and 42.6 ng/ml). The results of hormone assays indicated that bioavailability of steroid hormones remained relatively constant, regardless of the IVM system used. The plasticware with the addition of T resulted in significantly higher cleavage rates (80.0 ± 2.1%) than any other combination of treatments (plasticware × A₄: 71.5 ± 2.6%; glassware × T: 71.2 ± 1.9%; and glassware × A₄: 71.4 ± 2.4%). The blastocyst formation rate for the plasticware × T treatment (39.7 ± 2.5%) was significantly greater than for all other combinations (glassware × T: 28.7 ± 2.2%; glassware × A₄: 24.0 ± 2.8%; and plasticware × A₄: 19.8 ± 3.0%) and the low dose of T (37.1 ± 2.5%) resulted in higher (p < 0.05) blastocyst formation rates than all other treatments (T high dose: 29.2 ± 2.5%; A₄ high dose: 27.1 ± 2.9%; and A₄ low dose: 20.2 ± 3.0%). The proportion of male embryos was greater (p < 0.05) in plastic than glass dishes in the low-dose A₄ group (59.1 ± 8.7% vs. 38.2 ± 5.5%, plasticware vs. glassware, respectively) and it tended to be greater (p < 0.08) in the control groups and high-dose A₄ group, but not in the T groups. There was a moderate positive correlation between blastocyst formation rates across all treatment and control groups, and the percentage of male bovine embryos (r = 0.38, p < 0.05). In summary, specific combinations of androgen and glassware/plasticware treatments did alter early bovine embryo development and sex ratio. The addition of T to IVM media increased the cleavage and blastocyst formation rates in plasticware and may be employed to improve the efficiency of the standard in vitro embryo production systems. Androstenedione appeared to enhance whereas testosterone nullified the deviation in sex ratio (pro-femaleness) associated with the use of glass IVM dishes.     

Alkaloids from the roots of Stemona javanica (Kunth) Engl. (Stemonaceae) and their anti-malarial,acetylcholinesterase inhibitory and cytotoxic activities

Two new protostemonine-type alkaloids, javastemonine A and B (3 and 4) have been isolated from the root extracts of Stemona javanica together with four known Stemona alkaloids, 13-demethoxy-11(S*),12(R*)-dihydroprotostemonine (1), isoprotostemonine (2), protostemonine and isomaistemonine. The structures and relative configurations of the new alkaloids were determined by spectroscopic analysis. The alkaloids 1 and 2 and protostemonine showed moderated antiplasmodial activities against the Plasmodium falciparum strains, TM4 (IC₅₀ values of 17.7 ± 3.7, 16.8 ± 5.4, 16.0 ± 4.2 μg/mL, respectively) and K1 (IC₅₀ values of 16.8 ± 3.1, 14.1 ± 3.7, 11.9 ± 3.3 μg/mL, respectively). These compounds showed no significant cytotoxicities against KB or Vero cells or acetylcholinesterase inhibitory activities.     

Exenatide-loaded PLGA microspheres with improved glycemic control: In vitro bioactivity and in vivo pharmacokinetic profiles after subcutaneous administration to SD rats

A subcutaneous exenatide delivery system was developed and characterized in vitro and in vivo. The results clearly showed that the exenatide loaded PLGA microspheres prepared by using a non-aqueous processing medium had low burst release and high drug encapsulation efficiency. Exenatide loaded in the microspheres preserved its bioactivity. The pharmacokinetics parameters were determined after subcutaneous administration of microspheres to SD rats. The plasma concentration of the single dose of the sustained-release microspheres attained C_max of 108.19 ± 14.92 ng/ml at t_max of 1.33 ± 0.58 h and the t_1/2 was 120.65 ± 44.18 h. There was a linear correlation between the in vitro and in vivo release behavior (R² = 0.888). Exenatide loaded microspheres may prove to have great potential for clinical use.     

Syntheses of 4,6-dihydroxypyrimidine diones,their urease inhibition,in vitro,in silico,and kinetic studies

A library of 4,6-dihydroxypyrimidine diones (1–35) were synthesized and evaluated for their urease inhibitory activity. Structure-activity relationships, and mechanism of inhibition were also studied. All compounds were found to be active with IC₅₀ values between 22.6 ± 1.14–117.4 ± 0.73 µM, in comparison to standard, thiourea (IC₅₀ = 21.2 ± 1.3 µM). Kinetics studies on the most active compounds 2–7, 16, 17, 28, and 33 were performed to investigate their modes of inhibition, and dissociation constants K_i. Compounds 2, 3, 7, 16, 28, and 33 were found to be mixed-type of inhibitors with K_i values in the range of 7.91 ± 0.024–13.03 ± 0.013 µM, whereas, compounds 4–6, and 17 were found to be non-competitive inhibitors with K_i values in the range of 9.28 ± 0.019–13.05 ± 0.023 µM. In silico study was also performed, and a good correlation was observed between experimental and docking studies. This study is continuation of our previously reported urease inhibitory activity of pyrimidine diones, representing potential leads for further research as possible treatment of diseases caused by ureolytic bacteria.     

Radiological properties of 3D printed materials in kilovoltage and megavoltage photon beams

PurposeThis study evaluates the radiological properties of different 3D printing materials for a range of photon energies, including kV and MV CT imaging and MV radiotherapy beams.MethodsThe CT values of a number of materials were measured on an Aquilion One CT scanner at 80 kVp, 120 kVp and a Tomotherapy Hi Art MVCT imaging beam. Attenuation of the materials in a 6 MV radiotherapy beam was investigated.ResultsPlastic filaments printed with various infill densities have CT values of −743 ± 4, −580 ± 1 and −113 ± 3 in 120 kVp CT images which approximate the CT values of low-density lung, high-density lung and soft tissue respectively. Metal-infused plastic filaments printed with a 90% infill density have CT values of 658 ± 1 and 739 ± 6 in MVCT images which approximate the attenuation of cortical bone. The effective relative electron density RED_eff is used to describe the attenuation of a megavoltage treatment beam, taking into account effects relating to the atomic number and mass density of the material. Plastic filaments printed with a 90% infill density have RED_eff values of 1.02 ± 0.03 and 0.94 ± 0.02 which approximate the relative electron density RED of soft tissue. Printed resins have RED_eff values of 1.11 ± 0.03 and 1.09 ± 0.03 which approximate the RED of bone mineral.Conclusions3D printers can model a variety of body tissues which can be used to create phantoms useful for both imaging and dosimetric studies.     

Partial purification and kinetic characterization of mushroom stem polyphenoloxidase and determination of its storage stability in different lyophilized forms

Monophenolase (1011 ± 626 U/g AP) and diphenolase activities (5163 ± 3059 U/g AP) of PPO in acetone powders (APs) of different mushroom stems varied considerably. However, the limited variation of average dipenolase (L-DOPA) to monophenolase (L-tyrosine) activity ratio (5.4 ± 0.7) in crude extracts showed the homogeneity of PPO from different mushroom stems. The change in extraction material or partial purification method (ammonium sulfate or acetone precipitation) did not affect the temperature stability, temperature and pH dependency and K_m of monophenolase activity considerably. However, some changes were observed in pH stability and substrate specificity of PPO in different parties of mushroom stems. The most important aspects of mushroom stem PPO are its lower diphenolase to monophenolase activity ratio than mushroom cap PPO, low temperature dependency of activity between 25 and 40 °C (E_a = 30 kJ/mol), broad optimum pH between 6 and 8, but lack of activity pH ≤5, and ability to use phloridzin as substrate. The mushroom stem PPOs partially purified and lyophilized by using sucrose, dextran or alginate showed moderate to high stability at −18 °C for 6–6.5 months. Thus, the mushroom stems obtained as a waste material during mushroom processing may be used as a more homogenous source than whole mushrooms to obtain PPO used for different industrial, clinical or research purposes.     

Hepatic uptake and biliary excretion of manganese in the little skate,Leucoraja erinacea

The liver is a major organ involved in regulating whole body manganese (Mn) homeostasis; however, the mechanisms of Mn transport across the hepatocyte basolateral and canalicular membranes remain poorly defined. To gain insight into these transport steps, the present study measured hepatic uptake and biliary excretion of Mn in an evolutionarily primitive marine vertebrate, the elasmobranch Leucoraja erinacea, the little skate. Mn was rapidly removed from the recirculating perfusate of isolated perfused skate livers in a dose-dependent fashion; however, only a small fraction was released into bile (< 2% in 6 h). Mn was also rapidly taken up by freshly isolated skate hepatocytes in culture. Mn uptake was inhibited by a variety of divalent metals, but not by cesium. Analysis of the concentration-dependence of Mn uptake revealed of two components, with apparent K_m values 1.1 ± 0.1 µM and 112 ± 29 µM. The K_m value for the high-affinity component was similar to the measured skate blood Mn concentration, 1.9 ± 0.5 µM. Mn uptake was reduced by nearly half when bicarbonate was removed from the culture medium, but was unaffected by a change in pH from 6.5 to 8.5, or by substitution of Na with Li or K. Mn efflux from the hepatocytes was also rapid, and was inhibited when cells were treated with 0.5 mM 2,4-dinitrophenol to deplete ATP levels. These data indicate that skate liver has efficient mechanisms for removing Mn from the sinusoidal circulation, whereas overall biliary excretion is low and appears to be mediated in part by an ATP-sensitive mechanism.     

Active immunization against inhibin-based peptides to increase ovulation rate in non-prolific Malpura ewes

The aim of this study was to explore the possibility of increasing the ovulation rate of Malpura, a non-prolific tropical breed of sheep by immunization against inhibin-based peptide immunogens. Ewes were divided into three groups (n = 5 each) and actively immunized against the synthetic peptides from the α_C [bIα(1–29)-Tyr³⁰] or α_N [bI-₄₃-Tyr¹⁵²(153–167)Cys¹⁶⁸] area of the bovine inhibin α-subunit conjugated to ovalbumin or against ovalbumin (control). Each ewe received a primary immunization of 400 μg immunogen and 3 boosters, each of 200 μg immunogen at 4-week intervals. Estrus was synchronized using a double PGF₂α injection schedule and laparoscopy was performed after each estrus to determine the ovulation response. Immunization against both the peptides did not affect the interval from PGF treatment to the onset of estrus, the duration of estrus and the number of large unovulated follicles. In contrast to the complete absence of multiple ovulations in the controls, all the ewes immunized against α_C or α_N peptides showed multiple ovulations (range 2–7) in all the three estrous cycles evaluated, except for one ewe immunized against the α_N peptide, which exhibited multiple ovulations in only 1 out of the 3 estrous cycles. Compared to that of the controls (1.0 ± 0.9, 1.0 ± 0.0 and 0.6 ± 0.2, respectively), the mean ovulation rate was higher (P < 0.01) in the ewes immunized against the αC (4.8 ± 1.02, 5.0 ± 1.05 and 5.0 ± 0.45, respectively) or against αN (4.5 ± 1.19, 2.5 ± 0.87 and 2.7 ± 0.75, respectively, P < 0.05) peptide in estrous cycles numbers 1, 2 and 3. These results show that active immunization against inhibin-based peptide immunogens is effective in increasing ovulation rate in Malpura, a non-prolific breed of sheep and that it may be an alternative to conventional superovulation regimes.     

Synthesis and antioxidant evaluation of (S,S)- and (R,R)-secoisolariciresinol diglucosides (SDGs)

Secoisolariciresinol diglucosides (SDGs) (S,S)-SDG-1 (major isomer in flaxseed) and (R,R)-SDG-2 (minor isomer in flaxseed) were synthesized from vanillin via secoisolariciresinol (6) and glucosyl donor 7 through a concise route that involved chromatographic separation of diastereomeric diglucoside derivatives (S,S)-8 and (R,R)-9. Synthetic (S,S)-SDG-1 and (R,R)-SDG-2 exhibited potent antioxidant properties (EC₅₀ = 292.17 ± 27.71 μM and 331.94 ± 21.21 μM, respectively), which compared well with that of natural (S,S)-SDG-1 (EC₅₀ = 275.24 ± 13.15 μM). These values are significantly lower than those of ascorbic acid (EC₅₀ = 1129.32 ± 88.79 μM) and α-tocopherol (EC₅₀ = 944.62 ± 148.00 μM). Compounds (S,S)-SDG-1 and (R,R)-SDG-2 also demonstrated powerful scavenging activities against hydroxyl [natural (S,S)-SDG-1: 3.68 ± 0.27; synthetic (S,S)-SDG-1: 2.09 ± 0.16; synthetic (R,R)-SDG-2: 1.96 ± 0.27], peroxyl [natural (S,S)-SDG-1: 2.55 ± 0.11; synthetic (S,S)-SDG-1: 2.20 ± 0.10; synthetic (R,R)-SDG-2: 3.03 ± 0.04] and DPPH [natural (S,S)-SDG-1: EC₅₀ = 83.94 ± 2.80 μM; synthetic (S,S)-SDG-1: EC₅₀ = 157.54 ± 21.30 μM; synthetic (R,R)-SDG-2: EC₅₀ = 123.63 ± 8.67 μM] radicals. These results confirm previous studies with naturally occurring (S,S)-SDG-1 and establish both (S,S)-SDG-1 and (R,R)-SDG-2 as potent antioxidants and free radical scavengers for potential in vivo use.     

Algorithmic detection of the beginning and end of bipolar electrograms: Implications for novel methods to assess local activation time during atrial tachycardia

Activation mapping is required to effectively ablate atrial tachycardia (AT). Conventional tools to assess local activation time (LAT) are based upon the peak of the bipolar electrogram (B-EGM, LAT_Peak) and the maximal negative slope of the unipolar electrogram (U-EGM, LAT_Slope). Bipolar electrograms are influenced by wavefront direction, bipole orientation, and inter-electrode spacing causing ambiguity in peak detection, whereas unipolar electrograms are disturbed by the presence of far-field signals. We developed a new algorithm to detect the beginning and end of bipolar electrograms (t_begin and t_end). Then, we introduced new LAT methods related to the onset of B-EGMs (LAT_Onset), the center of mass of B-EGMs (LAT_CoM), and the slope of U-EGMs within a pre-defined window (LAT_Slope-hybrid).In total 3752 recordings from 31 AT patients were retrospectively analyzed. The signal-to-noise ratio (SNR) for B-EGMs was calculated to differentiate algorithmically high from low quality electrograms (HQ and LQ). In a subset of 328 B-EGMs, five experts validated the t_begin as determined by the algorithm by visual rating. The newly developed LAT methods were compared to the conventional LAT methods and to one another (Bland–Altman plots) in both HQ (n = 3003) and LQ EGMs (n = 749).The t_begin algorithm was accurate (deviation < ±10 ms) in 96 ± 4% of HQ and 91 ± 8% of LQ B-EGMs. BA plots revealed the following difference (bias) and variation in HQ and LQ EGMs respectively: (1) LAT_Onset vs. LAT_Peak: 27 ± 30 ms and 24 ± 62 ms; (2) LAT_CoM vs. LAT_Peak: 0 ± 16 ms and 2 ± 38 ms; (3) LAT_Slope-hybrid vs. LAT_Slope: 1 ± 32 ms and 15 ± 110 ms; (4) LAT_Onset vs. LAT_CoM: 22 ± 24 ms and 18 ± 22 ms; (5) LAT_Onset vs. LAT_Slope-hybrid: 16 ± 18 ms and 13 ± 22 ms; and (6) LAT_CoM vs. LAT_Slope-hybrid: 5 ± 20 ms and 4 ± 18 ms.In the present study, we introduced three new methods to assess local activation time in AT, based upon an algorithm detecting accurately the beginning and end of the B-EGM complex. BA analysis of the new methods showed similar variation in high and low quality EGMs, suggesting that they introduce less ambiguity than the conventional peak method. LAT_Onset consistently yielded an earlier activation moment. LAT_Slope-hybrid – by blanking far-field potentials – seems to be the optimal method for detection of the maximal negative slope in U-EGMs. Interestingly, LAT_CoM in B-EGMs coincided with the maximal negative slope in U-EGMs, suggesting its physiological sense and future use. The new LAT methods can be implemented in real-time mapping applications.     

Use of Saccharum spontaneum (wild sugarcane) as biomaterial for cell immobilization and modulated ethanol production by thermotolerant Saccharomyces cerevisiae VS3

Saccharum spontaneum is a wasteland weed consists of 45.10 ± 0.35% cellulose and 22.75 ± 0.28% of hemicellulose on dry solid (DS) basis. Aqueous ammonia delignified S. spontaneum yielded total reducing sugars, 53.91 ± 0.44 g/L (539.10 ± 0.55 mg/g of substrate) with a hydrolytic efficiency of 77.85 ± 0.45%. The enzymes required for hydrolysis were prepared from culture supernatants of Aspergillus oryzae MTCC 1846. A maximum of 0.85 ± 0.07 IU/mL of filter paperase (FPase), 1.25 ± 0.04 IU/mL of carboxy methyl cellulase (CMCase) and 55.56 ± 0.52 IU/mL of xylanase activity was obtained after 7 days of incubation at 28 ± 0.5 °C using delignified S. spontaneum as carbon source under submerged fermentation conditions. Enzymatic hydrolysate of S. spontaneum was then tested for ethanol production under batch and repeated batch production system using “in-situ” entrapped Saccharomyces cerevisiae VS₃ cells in S. spontaneum stalks (1 cm × 1 cm) size. Immobilization was confirmed by the scanning electron microscopy (SEM). Batch fermentation of VS₃ free cells and immobilized cells showed ethanol production, 19.45 ± 0.55 g/L (yield, 0.410 ± 0.010 g/g) and 21.66 ± 0.62 g/L (yield, 0.434 ± 0.021 g/g), respectively. Immobilized VS₃ cells showed maximum ethanol production (22.85 ± 0.44 g/L, yield, 0.45 ± 0.04 g/g) up to 8th cycle during repeated batch fermentation followed by a gradual reduction in subsequent cycles of fermentation.     

Dosimetric comparison of RapidPlan and manually optimized plans in volumetric modulated arc therapy for prostate cancer

PurposeThis study evaluated whether RapidPlan based plans (RP plans) created by a single optimization, are usable in volumetric modulated arc therapy (VMAT) for patients with prostate cancer.MethodsWe used 51 previously administered VMAT plans to train a RP model. Thirty RP plans were created by a single optimization without planner intervention during optimization. Differences between RP plans and clinical manual optimization (CMO) plans created by an experienced planner for the same patients were analyzed (Wilcoxon tests) in terms of homogeneity index (HI), conformation number (CN), D_95%, and D_2% to planning target volume (PTV), mean dose, V_50Gy, V_70Gy, V_75Gy, and V_78Gy to rectum and bladder, monitor unit (MU), and multi-leaf collimator (MLC) sequence complexity.ResultsRP and CMO values for PTV D_95%, PTV D_2%, HI, and CN were significantly similar (p < 0.05 for all). RP mean dose, V_50Gy, and V_70Gy to rectum were superior or comparable to CMO values; RP V_75Gy and V_78Gy were higher than in CMO plans (p < 0.05). RP bladder dose-volume parameter values (except V_78Gy) were lower than in CMO plans (p < 0.05). MU values were RP: 730 ± 55 MU and CMO: 580 ± 37 MU (p < 0.05); and MLC sequence complexity scores were RP: 0.25 ± 0.02 and CMO: 0.35 ± 0.03 (p < 0.05).ConclusionsRP plans created by a single optimization were clinically acceptable in VMAT for patient with prostate cancer. Our simple model could reduce optimization time, independently of planner’s skill and knowledge.     

Inhibition of serine and proline racemases by substrate-product analogues

d-Amino acids can play important roles as specific biosynthetic building blocks required by organisms or act as regulatory molecules. Consequently, amino acid racemases that catalyze the formation of d-amino acids are potential therapeutic targets. Serine racemase catalyzes the reversible formation of d-serine (a modulator of neurotransmission) from l-serine, while proline racemase (an essential enzymatic and mitogenic protein in trypanosomes) catalyzes the reversible conversion of l-proline to d-proline. We show the substrate-product analogue α-(hydroxymethyl)serine is a modest, linear mixed-type inhibitor of serine racemase from Schizosaccharomyces pombe (K_i = 167 ± 21 mM, K_i′ = 661 ± 81 mM, cf. K_m = 19 ± 2 mM). The bicyclic substrate-product analogue of proline, 7-azabicyclo[2.2.1]heptan-7-ium-1-carboxylate is a weak inhibitor of proline racemase from Clostridium sticklandii, giving only 29% inhibition at 142.5 mM. However, the more flexible bicyclic substrate-product analogue tetrahydro-1H-pyrrolizine-7a(5H)-carboxylate is a noncompetitive inhibitor of proline racemase from C. sticklandii (K_i = 111 ± 15 mM, cf. K_m = 5.7 ± 0.5 mM). These results suggest that substrate-product analogue inhibitors of racemases may only be effective when the active site is capacious and/or plastic, or when the inhibitor is sufficiently flexible.