Nitrogen utilisation,energy utilisation and methane emissions of sheep grazing in two types of pasture

Livestock grazing plays a significant role in maintaining grasslands and promoting animal production globally. To understand the livestock performance in sown pasture (SP) vs native pasture (NP) is important to ensure more effective grassland-livestock interactions with minimal environmental impact. A 2 (treatment) * 2 (period) Latin Square design experiment was conducted with 10 growing Hu sheep ♂ × thin-tailed Han sheep ♀ rams grazed perennially SP vs NP in an inland arid region of China. The objectives were to evaluate the effects of grazing management on nutrient digestibility, nitrogen (N) and energy utilisation and methane (CH₄) emission. The N intake, N retained and energy intake (gross energy (GE), and digestible and metabolisable energy) of sheep grazing in SP were significantly increased compared with those grazing in NP. There were significant linear relationships between DM intake (DMI) (g/kg BW or g/kg BW^0.75) or CH₄ (g/kg BW or g/kg BW^0.75) emissions and forage nutrient and GE concentrations within each grassland type. The linear regression analysis indicated that forage CP or ether extract concentration was a good predictor for DMI (g/kg BW or g/kg BW^0.75) (R² = 0.756 or 0.752), and CH₄ emission could be predicted using forage nutrient and GE concentrations (R² = 0.381–0.503). These results suggest that DMI and CH₄ emissions per unit metabolic BW were accurately predicted by multiple-factor combinations of forage nutrients, including ether extract and CP paired with GE. The present output could provide useful information for the development of sustainable sheep grazing systems in the inland arid regions of the world.     

The effect of hessian and straw as nesting materials on sow behaviour and piglet survival and growth to weaning

Sows are strongly driven to build a nest prior to farrowing, and the performance of this behaviour is linked to the environment in which the animal is housed. The aim of this study was to investigate the impact of two nest-building materials, hessian and straw, on peri-parturient sow behaviour, plasma cortisol concentration and piglet survival and performance in farrowing crates. In the first experiment, sows (parity 1.7 ± 0.1) were allocated to four treatments: (n = 15), straw provided in the lead up to farrowing in an open farrowing pen, with the pen closed after farrowing (STRAW OPEN); (n = 14), straw provided in the lead up to farrowing in a closed farrowing pen (STRAW CLOSED); (n = 15), a closed farrowing pen with hessian sacks provided in the lead up to farrowing (HESSIAN) and; (n = 13), a closed farrowing pen with no nesting materials provided (CONTROL). A second experiment was performed on a separate farm to assess the effect of the same four treatments were applied to sows (parity 2.9 ± 0.1): SRAW OPEN (n = 68), STRAW CLOSED (n = 64), HESSIAN (n = 66) and CONTROL (n = 66), at a commercial level. The first experiment revealed that providing conventionally housed sows with straw or hessian in the lead up to parturition stimulated sows to perform nest-building behaviours similar to sows housed in an open pen with access to straw (nosing events; 16 ± 11 (CONTROL); 169 ± 36 (HESSIAN); 118 ± 29 (STRAW CLOSED); 199 ± 53 (STRAW OPEN); P < 0.05). Additionally, crated sows provided with straw had reduced cortisol levels immediately after farrowing compared to all other treatments (21.9 ± 6.1 ng/ml vs CONTROL; 49.3 ± 8.6 ng/ml; P < 0.01). Piglets born to STRAW CLOSED sows displayed the highest colostrum intake levels (404.8 ± 22.7 g vs CONTROL 361.9 ± 21.9 g; P < 0.01). The second experiment demonstrated a reduced incidence of piglet mortality both prior to fostering (0.7 ± 0.2; P = 0.001) and after fostering (0.7 ± 0.2; P = 0.001) in litters born to sows which were housed in conventional farrowing crates and provided with straw compared to CONTROL (prior to fostering 1.3 ± 0.2, and postfostering 1.1 ± 0.2). In conclusion, straw and hessian sacks are a suitable substrate for stimulating sows to exhibit nest-building behaviour under crated conditions. However, only the provision of straw in the crate environment improved piglet survival and positively affected sow welfare.     

Pasture dry matter intake per cow in intensive dairy production systems: effects of grazing and feeding management

The competitiveness and sustainability of low input cost dairy production systems are generally supported by efficient use of pasture in the diets. Therefore, pasture intake directly affects overall efficiency of these systems. We aimed to assess feeding and grazing management main factors that affect pasture dry matter intake (DMI) in commercial dairy farms during the different seasons of the year. Fortnightly visits to 28 commercial dairies were carried out between June 2016 and May 2017 to record production and price, supplement offered and price, pasture access time (PAT), herbage mass (HM) and allowance (HA). Only farms with the most contrasting estimated pasture DMI per cow (eDMI) were compared as systems with high (HPI; N = 8) or low (LPI; N = 8) pasture DMI. Despite a lower individual milk production in HPI than LPI (19.0 v. 23.3 ± 0.7 l/cow, P < 0.01), daily margin over feeding cost was not different between groups (3.07 v. 2.93 ± 0.15 U$S/cow for HPI and LPI, respectively). During autumn and winter, HPI cows ingested more pasture than LPI cows (8.3 v. 4.6 and 5.9 v. 2.9 ± 0.55 kg DM/cow per day, respectively, P < 0.01) although PAT, HM and HA were similar between groups. Both groups offered high supplementation levels during these seasons, even though greater in LPI than HPI (14.7 v. 9.7 ± 0.7 kg DM supplement/cow per day, respectively, P < 0.01). On the other hand, differences between groups for both pasture and supplement DMI were more contrasting during spring and summer (13.1 v. 7.3 ± 0.5 and 4.0 v. 11.4 ± 0.4 kg DM/cow per day for HPI and LPI, respectively, P < 0.01), with higher PAT in both seasons (P < 0.05) and higher HA during summer in HPI than LPI (P < 0.01). Unlike LPI, during these seasons HPI adjusted offered supplement according to HA, achieving a higher pasture eDMI and making more efficient use of available pastoral resource than LPI. As there was no grazing limiting condition for pasture harvesting in either group, the main factor affecting pasture DMI was a pasture by supplement substitution effect. These results reinforce the importance of an efficient grazing management, and using supplements to nutritionally complement pasture intake rather than as a direct way to increase milk production.     

The periparturient rise in fecal egg counts in three strains of Florida native ewes and its value in predicting resistance of lambs to Haemonchus contortus

The periparturient rise in fecal egg counts in three strains of Florida Native ewes and its value in predicting resistance of lambs to Haemonchus contortus. International Journal for Parasitology16: 185–189. Significant (P < 0.05) differences in the magnitude of the periparturient rise in fecal egg counts (PPR) occurred in ewes of three strains of Florida Native sheep. Non-lambing ewes (n = 12) and lactating University strain ewes (n = 31), a strain selected over a 26-year period for parasite resistance by survival at pasture without anthelmintic treatment, had lower fecal egg counts than ewes of either Maxcy strain (n= 17), a commercial strain treated regularly with anthelmintics, or Backlinie strain (n = 16), a strain of Florida Native sheep that had some crossbreeding to Hampshire and Suffolk in their genotype and were regularly treated with anthelmintics. Whereas both Maxcy and Backlinie ewes showed a pronounced PPR, none occurred in non-lambing ewes (n=12) and the slight rise in fecal egg counts observed in University ewes was not significantly different from that of the non-lambing ewes. Backlinie ewes having twin lambs (n = 5) had a higher PPR than Backlinie ewes with single lambs (n = 11) or Maxcy ewes with single lambs (n = 15), but all three groups had higher PPRs than University ewes with single lambs (n = 27). No differences occurred in fecal egg counts of non-lambing ewes regardless of strain. Six weeks after weaning fecal egg counts of all ewes were uniformly low regardless of strain or prior lactation status.Ram lambs born to these ewes showed no significant strain differences in Haemonchus contortus burdens after experimental infection, deworming with levamisole and reinfection, although University lambs (n= 13) had lowest mean worm burdens at necropsy (2437) followed by Maxcy lambs (n= 12, mean =3106) and Backlinie lambs (n= 13, mean =3670). There was no significant correlation between worm burdens in these lambs and the magnitude of the PPR in their dams. Eight to 16 weeks after turn out to a common parasite-infested pasture ewe lambs (n = 13) born to University ewes had lower fecal egg counts than similar ewe lambs (n=8) born to Backlinie ewes. Prior to this time differences were not significant and egg counts were generally low. The magnitude of the PPR in their dams was not significantly correlated with fecal egg counts or PCVs of individual ewe lambs.     

Syntheses,in vitro α-amylase and α-glucosidase dual inhibitory activities of 4-amino-1,2,4-triazole derivatives their molecular docking and kinetic studies

Thirty-three 4-amino-1,2,4-triazole derivatives 1–33 were synthesized by reacting 4-amino-1,2,4-triazole with a variety of benzaldehydes. The synthetic molecules were characterized via ¹H NMR and EI-MS spectroscopic techniques and evaluated for their anti-hyperglycemic potential. Compounds 1–33 exhibited good to moderate in vitro α-amylase and α-glucosidase inhibitory activities in the range of IC₅₀ values 2.01 ± 0.03–6.44 ± 0.16 and 2.09 ± 0.08–6.54 ± 0.10 µM as compared to the standard acarbose (IC₅₀ = 1.92 ± 0.17 µM) and (IC₅₀ = 1.99 ± 0.07 µM), respectively. The limited structure-activity relationship suggested that different substitutions on aryl part of the synthetic compounds are responsible for variable activity. Kinetic study predicted that compounds 1–33 followed mixed and non-competitive type of inhibitions against α-amylase and α-glucosidase enzymes, respectively. In silico studies revealed that both triazole and aryl ring along with different substitutions were playing an important role in the binding interactions of inhibitors within the enzyme pocket. The synthetic molecules were found to have dual inhibitory potential against both enzymes thus they may serve as lead candidates for the drug development and research in the future studies.     

Synthesis of alpha amylase inhibitors based on privileged indole scaffold

Twenty five derivatives of indole carbohydrazide (1–25) had been synthesized. These compounds were characterized using ¹H NMR and EI-MS, and further evaluated for their α-amylase inhibitory potential. The analogs (1–25) showed varying degree of α-amylase inhibitory potential.ranging between 9.28 and 599.0 µM when compared with standard acarbose having IC₅₀ value 8.78 ± 0.16 µM. Six analogs, 25 (IC₅₀ = 9.28 ± 0.153 µM), 22 (IC₅₀ = 9.79 ± 0.43 µM), 4 (IC₅₀ = 11.08 ± 0.357 µM), 1 (IC₅₀ = 12.65 ± 0.169 µM), 8 (IC₅₀ = 21.37 ± 0.07 µM) and 14 (IC₅₀ = 43.21 ± 0.14 µM) showed potent α-amylase inhibition as compared to the standard acarbose (IC₅₀ = 8.78 ± 0.16 µM). All other analogs displayed good to moderate inhibitory potential. Structure-activity relationship was established through the interaction of the active compounds with enzyme active site with the help of docking studies.     

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.     

Time course determination of the effects of rapid and gradual cooling after acute hyperthermia on body temperature and intestinal integrity in pigs

Rapid cooling after acute hyperthermia may cause a sustained increase in body temperature and exacerbate intestinal damage in pigs. Therefore, the study objective was to evaluate the temporal effects of rapid and gradual cooling on body temperature response and intestinal integrity after acute hyperthermia in pigs. In three repetitions, 54 pigs [83.3 ± 6.7 kg initial body weight (BW)], balanced by sex were exposed to thermoneutral conditions for 6 h (TN; n = 6 pigs/repetition; 21.1 ± 2.0°C), or heat stress conditions (HS; 39.3 ± 1.6°C) for 3 h, followed by a 3 h recovery period of gradual cooling [HSGC; n = 6 pigs/repetition; gradual decrease from HS to TN conditions] or rapid cooling [HSRC; n = 6 pigs/repetition; rapid TN exposure and cold water (4.0°C) dousing every 30 min for 1.5 h]. Feed was withheld throughout the entire 6 h period, but water was provided ad libitum. Gastrointestinal (T_GI) and rectal (T_R) temperatures were recorded every 15 min during the HS and recovery periods. Six pigs per repetition (n = 2/treatment) were euthanized and jejunal and ileal samples were collected for histology immediately after (d 0), 2 d after, and 4 d after the recovery period. Data were analyzed using PROC MIXED in SAS 9.4. Overall, rapid cooling reduced T_R and T_GI (P < 0.01; 0.95°C and 0.74°C, respectively) compared to gradual cooling. Jejunal villus height was reduced overall (P = 0.02; 14.01%) in HSGC compared to HSRC and TN pigs. Jejunal villus height-to-crypt depth ratio was reduced overall (P = 0.05; 16.76%) in HSGC compared to TN pigs. Ileal villus height was reduced overall (P < 0.01; 16.95%) in HSGC compared to HSRC and TN pigs. No other intestinal morphology differences were detected. In summary, HSRC did not cause a sustained increase in body temperature and did not negatively impact biomarkers of intestinal integrity in pigs.     

Chemical constituents of Mussaenda erythrophylla Schumach. & Thonn. (Rubiaceae) and their chemophenetic significance

The chemical investigation of the CH₂Cl₂/MeOH (1:1) extract from the aerial part of Mussaenda erythrophylla Schumach. & Thonn. (Rubiaceae) resulted in the isolation of sixteen known compounds (1–16) distributed in coumarins, flavonoid glucosides, quinic acid derivatives, triterpenoids, monoglycerid, steroids, tetraterpenoid and polyol. The structures of the compounds were determined by spectrometric and spectroscopic analysis including MS and NMR data followed by their comparison with reported ones in the literature. The chemophenetic significance of the isolated compounds was discussed. The crude extract and some of the isolated compounds were assessed in vitro for their antileishmanial, cytotoxic and antiplasmodial activities. The crude extract of M. erythrophylla showed moderate antileishmanial activity (IC₅₀ = 61.6 μg/mL) while the hexane soluble fraction showed good antileishmanial activity (IC₅₀ = 31.06 μg/mL) compared to the reference drug amphotericin B (IC₅₀ = 0.11 μM). Compounds 11 and 9 also exhibited potent antileishmanial activity (IC₅₀ = 53.7–52.0 μM). The crude extract as well as the ethyl acetate soluble fraction also exhibited good antiplasmodial activity (IC₅₀ = 7.43 ± 0.00 μg/mL and 14.49 ± 2.96 μg/mL respectively), while compounds 11, 15 and 16 showed weak activity with IC₅₀ > 20 μM compared to the reference drug artemisinin (IC₅₀ = 0.014 ± 0.001 μM).     

Serum progesterone concentrations,follicular development and time of ovulation using a new progesterone releasing device (DICO®) in sheep

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 < 0.05). However, the re-used DICO^® 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.     

Synthesis,in vitro α-glucosidase inhibitory activity and molecular docking studies of new thiazole derivatives

Current study based on the synthesis of new thiazole derivatives via “one pot” multicomponent reaction, evaluation of their in vitro α-glucosidase inhibitory activities, and in silico studies. All synthetic compounds were fully characterized by ¹H NMR, ¹³C NMR and EIMS. CHN analysis was also performed. These newly synthesized compounds showed activities in the range of IC₅₀ = 9.06 ± 0.10–82.50 ± 1.70 μM as compared to standard acarbose (IC₅₀ = 38.25 ± 0.12 μM). It is worth mentioning that most of the compounds such as 1 (IC₅₀ = 23.60 ± 0.39 μM), 2 (IC₅₀ = 22.70 ± 0.60 μM), 3 (IC₅₀ = 22.40 ± 0.32 μM), 4 (IC₅₀ = 26.5 ± 0.40 μM), 6 (IC₅₀ = 34.60 ± 0.60 μM), 7 (IC₅₀ = 26.20 ± 0.43 μM), 8 (IC₅₀ = 14.06 ± 0.18 μM), 9 (IC₅₀ = 17.60 ± 0.28 μM), 10 (IC₅₀ = 27.16 ± 0.41 μM), 11 (IC₅₀ = 19.16 ± 0.19 μM), 12 (IC₅₀ = 9.06 ± 0.10 μM), 13 (IC₅₀ = 12.80 ± 0.21 μM), 14 (IC₅₀ = 11.94 ± 0.18 μM), 15 (IC₅₀ = 16.90 ± 0.20 μM), 16 (IC₅₀ = 12.60 ± 0.14 μM), 17 (IC₅₀ = 16.30 ± 0.29 μM), and 18 (IC₅₀ = 32.60 ± 0.61 μM) exhibited potent inhibitory potential. Molecular docking study was performed in order to understand the molecular interactions between the molecule and enzyme. Newly identified α-glucosidase inhibitors except few were found to be completely non-toxic.     

Indole acrylonitriles as potential anti-hyperglycemic agents: Synthesis, α-glucosidase inhibitory activity and molecular docking studies

One of the most prevailing metabolic disorder diabetes mellitus has become the global health issue that has to be addressed and cured. Different marketed drugs have been made available for the treatment of diabetes but there is still a need of introducing new therapeutic agents that are economical and have lesser or no side effects. The current study deals with the synthesis of indole acrylonitriles (3–23) and the evaluation of these compounds for their potential for α-glucosidase inhibition. The structures of these synthetic molecules were deduced by using different spectroscopic techniques. Acarbose (IC₅₀ = 2.91 ± 0.02 μM) was used as standard in this study and the synthetic molecules (3–23) have shown promising α-glucosidase inhibitory activity. Compounds 4, 8, 10, 11, 14, 18, and 21 displayed superior inhibition of α-glucosidase enzyme in the range of (IC₅₀ = 0.53 ± 0.01–1.36 ± 0.04 μM) as compared to the standard acarbose. Compound 10 (IC₅₀ = 0.53 ± 0.01 μM) was the most effective inhibitor of this library and displayed many folds enhanced activity in contrast to the standard. Molecular docking of synthetic compounds was performed to verify the binding interactions of ligand with the active site of enzyme. This study had identified a number of potential α-glucosidase inhibitors that can be used for further research to identify a potent therapeutic agent against diabetes.     

Identification of new potent inhibitor of aldose reductase from Ocimum basilicum

Recent efforts to develop cure for chronic diabetic complications have led to the discovery of potent inhibitors against aldose reductase (AKR1B1, EC 1.1.1.21) whose role in diabetes is well-evident. In the present work, two new natural products were isolated from the ariel part of Ocimum basilicum; 7-(3-hydroxypropyl)-3-methyl-8-β-O-d-glucoside-2H-chromen-2-one (1) and E-4-(6′-hydroxyhex-3′-en-1-yl)phenyl propionate (2) and confirmed their structures with different spectroscopic techniques including NMR spectroscopy etc. The isolated compounds (1, 2) were evaluated for in vitro inhibitory activity against aldose reductase (AKR1B1) and aldehyde reductase (AKR1A1). The natural product (1) showed better inhibitory activity for AKR1B1 with IC₅₀ value of 2.095 ± 0.77 µM compare to standard sorbinil (IC₅₀ = 3.14 ± 0.02 µM). Moreover, the compound (1) also showed multifolds higher activity (IC₅₀ = 0.783 ± 0.07 µM) against AKR1A1 as compared to standard valproic acid (IC₅₀ = 57.4 ± 0.89 µM). However, the natural product (2) showed slightly lower activity for AKR1B1 (IC₅₀ = 4.324 ± 1.25 µM). Moreover, the molecular docking studies of the potent inhibitors were also performed to identify the putative binding modes within the active site of aldose/aldehyde reductases.     

Synthesis of thiobarbituric acid derivatives: In vitro α-glucosidase inhibition and molecular docking studies

Synthesis, structure, and evaluation of in vitro α-glucosidase enzyme inhibition of a new class of diethylammonium salts of aryl substituted thiobarbituric acid is described. This protocol is straight, environmentally benign and efficient, involving Aldol-Michael addition reaction in one pot fashion. The 3D chemical structures of the synthesized compounds were assigned based on spectroscopic methods and X-ray single crystal diffraction analyses. All synthesized compounds 3a-3n were evaluated for their in vitro α-glucosidase enzyme inhibitory activity, whereas acarbose was used as the standard drug (IC₅₀ = 840 ± 1.73 µM). All tested compounds were found to possess varying degree of α-glucosidase enzyme inhibition activity with (IC₅₀ = 19.46 ± 1.84–415.8 ± 4.0 µM). Compound 3i (IC₅₀ = 19.4 ± 1.84 µM) exhibited the highest activity. To the best of knowledge this is the first report of the in vitro α-glucosidase enzyme inhibition by the diethylamonium salts of aryl substituted thiobarbituric acid. Furthermore, molecular docking studies of selected compounds were also performed to see interactions between active compounds and binding sites.     

Regulation of basal and norepinephrine-induced cAMP and ICa in hiPSC-cardiomyocytes: Effects of culture conditions and comparison to adult human atrial cardiomyocytes

BackgroundThere is ongoing interest in generating cardiomyocytes derived from human induced pluripotent stem cells (hiPSC) to study human cardiac physiology and pathophysiology. Recently we found that norepinephrine-stimulated calcium currents (I_Ca) in hiPSC-cardiomyocytes were smaller in conventional monolayers (ML) than in engineered heart tissue (EHT). In order to elucidate culture specific regulation of β₁-adrenoceptor (β₁-AR) responses we investigated whether action of phosphodiesterases (PDEs) may limit norepinephrine effects on I_Ca and on cytosolic cAMP in hiPSC-cardiomyocytes. Results were compared to adult human atrial cardiomyocytes.MethodsAdult human atrial cardiomyocytes were isolated from tissue samples obtained during open heart surgery. All patients were in sinus rhythm. HiPSC-cardiomyocytes were dissociated from ML and EHT. Förster-resonance energy transfer (FRET) was used to monitor cytosolic cAMP (Epac1-camps sensor, transfected by adenovirus). I_Ca was recorded by whole-cell patch clamp technique. Cilostamide (300 nM) and rolipram (10 μM) were used to inhibit PDE3 and PDE4, respectively. β₁-AR were stimulated with the physiological agonist norepinephrine (100 μM).ResultsIn adult human atrial cardiomyocytes, norepinephrine increased cytosolic cAMP FRET ratio by +13.7 ± 1.5% (n = 10/9, mean ± SEM, number of cells/number patients) and I_Ca by +10.4 ± 1.5 pA/pF (n = 15/10). This effect was not further increased in the concomitant presence of rolipram, cilostamide and norepinephrine, indicating saturation by norepinephrine alone. In ML hiPSC-cardiomyocytes, norepinephrine exerted smaller increases in cytosolic cAMP and I_Ca (FRET +9.6 ± 0.5% n = 52/21, number of cells/number of ML or EHT, and I_Ca + 1.4 ± 0.2 pA/pF n = 34/7, p < 0.05 each) and both were augmented in the presence of the PDE4 inhibitor rolipram (FRET +16.7 ± 0.8% n = 94/26 and I_Ca + 5.6 ± 1.4 pA/pF n = 11/5, p < 0.05 each). Cilostamide increased the response to norepinephrine on FRET (+12.7 ± 0.5% n = 91/19, p < 0.05), but not on I_Ca. In EHT hiPSC-cardiomyocytes, norepinephrine responses on both, FRET and I_Ca, were larger than in ML (FRET +12.1 ± 0.3% n = 87/32 and I_Ca + 3.3 ± 0.2 pA/pF n = 13/5, p < 0.05 each). Rolipram augmented the norepinephrine effect on I_Ca (+6.2 ± 1.6 pA/pF; p < 0.05 vs. norepinephrine alone, n = 10/4), but not on FRET.ConclusionOur results show culture-dependent differences in hiPSC-cardiomyocytes. In conventional ML but not in EHT, maximum norepinephrine effects on cytosolic cAMP depend on PDE3 and PDE4, suggesting immaturity when compared to the situation in adult human atrial cardiomyocytes. The smaller I_Ca responses to norepinephrine in ML and EHT vs. adult human atrial cardiomyocytes depend at least partially on a non-physiological large impact of PDE4 in hiPSC-cardiomyocytes.     

5-Bromo-2-aryl benzimidazole derivatives as non-cytotoxic potential dual inhibitors of α-glucosidase and urease enzymes

On the basis of previous report on promising α-glucosidase inhibitory activity of 5-bromo-2-aryl benzimidazole derivatives, these derivatives were further screened for urease inhibitory and cytotoxicity activity in order to get more potent and non-cytotoxic potential dual inhibitor for the patients suffering from diabetes as well as peptic ulcer. In this study, all compounds showed varying degree of potency in the range of (IC₅₀ = 8.15 ± 0.03–354.67 ± 0.19 μM) as compared to standard thiourea (IC₅₀ = 21.25 ± 0.15 μM). It is worth mentioning that derivatives 7 (IC₅₀ = 12.07 ± 0.05 μM), 8 (IC₅₀ = 10.57 ± 0.12 μM), 11 (IC₅₀ = 13.76 ± 0.02 μM), 14 (IC₅₀ = 15.70 ± 0.12 μM) and 22 (IC₅₀ = 8.15 ± 0.03 μM) were found to be more potent inhibitors than standard. All compounds were also evaluated for cytotoxicity towards 3T3 mouse fibroblast cell line and found to be completely non-toxic. Previously benzimidazole 1–25 were also showed α-glucosidase inhibitory potential. In silico studies were performed on the lead molecules i.e. 2, 7, 8, 11, 14, and 22, in order to rationalize the binding interaction of compounds with the active site of urease enzyme.     

The Added and Interpretative Value of CGM-Derived Parameters in Type 1 Diabetes Depends on the Level of Glycemic Control

ObjectiveIn type 1 diabetes mellitus (T1DM) management, continuous glucose monitoring (CGM)-derived parameters can provide additional insights, with time in range (TIR) and other parameters reflecting glycemic control and variability being put forward. This study aimed to examine the added and interpretative value of the CGM-derived indices TIR and coefficient of variation (CV%) in T1DM patients stratified according to their level of glycemic control by means of HbA1C.MethodsT1DM patients with a minimum disease duration of 10 years and without known macrovascular disease were enrolled. Patients were equipped with a blinded CGM device for 7 days. TIR and time spent in hypoglycemia and hyperglycemia were determined, and CV% was used as a parameter for glycemic variability. Pearson (r) and Spearman correlations (r_s) and a regression analysis were used to examine associations.ResultsNinety-five patients (age: 45 ± 10 years; HbA1C level: 7.7% ± 0.8% [61 ± 7 mmol/mol]) were included (mean blood glucose [MBG]: 159 ± 31 mg/dL; TIR: 55.8% ± 14.9%; CV%: 43.5% ± 7.8%) and labeled as having good (HbA1C level ≤7% [≤53 mmol/mol]; n = 20), moderate (7%-8%; n = 44), or poor (>8% [>64 mmol/mol]; n = 31) glycemic control. HbA1C was significantly associated with MBG (r_s = 0.48, P < .001) and time spent in hyperglycemia (total: r_s = 0.52; level 2: r = 0.46; P < .001) but not with time spent in hypoglycemia and CV%, even after an analysis of the HbA1C subgroups. Similarly, TIR was negatively associated with HbA1C (r = −0.53; P < .001), MBG (r_s = −0.81; P < .001), and time spent in hyperglycemia (total: r_s = −0.90; level 2: r_s = −0.84; P < .001) but not with time in hypoglycemia. The subgroup analyses, however, showed that TIR was associated with shorter time spent in level-2 hypoglycemia in patients with good (r_s = −0.60; P = .007) and moderate (r_s = −0.25; P = .047) glycemic control. In contrast, CV% was strongly positively associated with time in hypoglycemia (total: r_s = 0.78; level 2: r_s = 0.76; P < .001) but not with TIR or time in hyperglycemia in the entire cohort, although the subgroup analyses showed that TIR was negatively associated with CV% in patients with good glycemic control (r = −0.81, P < .001) and positively associated in patients with poor glycemic control (r = +0.47; P < .01).ConclusionThe CGM-derived metrics TIR and CV% are related to clinically important situations, TIR being strongly dependent on hyperglycemia and CV% being reflective of hypoglycemic risk. However, the interpretation and applicability of TIR and CV% and their relationship depends on the level of glycemic control of the individual patient, with CV% generally adding less clinically relevant information in those with poor control. This illustrates the need for further research and evaluation of composite measures of glycemic control in T1DM.     

The continuous presence of ewes in estrus in spring influences testicular volume,testicular echogenicity and testosterone concentration,but not LH pulsatility in rams

The continuous presence of active male small ruminants prevents seasonal anestrus in females, but evidence of the same mechanism operating from the females to the males is scarce. This study assessed the effects of the continuous presence of ewes in estrus in spring on ram sexual activity, testicular size and echogenicity, and LH and testosterone concentrations. On 1 March, 20 rams were assigned to two groups (n = 10 each): isolated (ISO) from other sheep, or stimulated (STI) by 12 ewes, which were separated from the rams by an openwork metal barrier, allowing contact between sexes. Each week, four ewes were induced into estrus by intravaginal sponges. Live weight, scrotal circumference, testicular width (TW) and length (TL) were recorded at the beginning and at the end of the experiment, and testicular volume (TV) was calculated; at the same time, testicular ultrasonography and color Doppler scanning were performed. Blood samples (March to May) were collected once per week for testosterone determinations, and at the end of the experiment, blood samples were collected for 6 h at 20-min intervals for LH analysis. Rams were exposed to four estrous ewes in a serving-capacity test. Scrotal circumference, TW and TL were higher in the STI than in the ISO rams (P < 0.05) in May, and TV was higher (P < 0.05) in the STI (391 ± 17 cm³) than in the ISO rams (354 ± 24 cm³). In ISO rams, the number of white pixels was higher (P < 0.01) in May (348 ± 74) than in March (94 ± 21) and differed significantly (P < 0.01) from that of the STI rams in May (160 ± 33). In ISO rams, the number of grey pixels was higher (P < 0.05) in May (107 ± 3) than it was in March (99 ± 1). Stimulated and ISO rams did not differ significantly in mean LH plasma concentrations (0.8 ± 0.5 v. 0.9 ± 0.4 ng/ml), LH pulses (2.1 ± 0.5 v. 2.2 ± 0.2) and amplitude (2.0 ± 0.4 v. 3.2 ± 0.7 ng/ml, respectively). Stimulated rams had significantly higher testosterone concentrations than ISO rams from April to the end of the experiment. Stimulated rams performed more (P < 0.05) mountings with intromission (3.0 ± 0.4) than did ISO rams (1.5 ± 0.5). In conclusion, after 3 months in the continuous presence of ewes in estrus in spring, rams had higher TV and some testicular echogenic parameters were modified than isolated rams. Although exposed rams also had higher levels of testosterone after 2 months in the presence of estrous ewes, their LH pulsatility at the end of the study was not modified.     

Dihydropyrano [2,3-c] pyrazole: Novel in vitro inhibitors of yeast α-glucosidase

Inhibition of α-glucosidase enzyme activity is a reliable approach towards controlling post-prandial hyperglycemia associated risk factors. During the current study, a series of dihydropyrano[2,3-c] pyrazoles (1–35) were synthesized and evaluated for their α-glucosidase inhibitory activity. Compounds 1, 4, 22, 30, and 33 were found to be the potent inhibitors of the yeast α-glucosidase enzyme. Mechanistic studies on most potent compounds reveled that 1, 4, and 30 were non-competitive inhibitors (K_i = 9.75 ± 0.07, 46 ± 0.0001, and 69.16 ± 0.01 μM, respectively), compound 22 is a competitive inhibitor (K_i = 190 ± 0.016 μM), while 33 was an uncompetitive inhibitor (K_i = 45 ± 0.0014 μM) of the enzyme. Finally, the cytotoxicity of potent compounds (i.e. compounds 1, 4, 22, 30, and 33) was also evaluated against mouse fibroblast 3T3 cell line assay, and no toxicity was observed. This study identifies non-cytotoxic novel inhibitors of α-glucosidase enzyme for further investigation as anti-diabetic agents.     

Effect of willow supplementation upon plasma amino acid concentration in ewes grazing drought pastures of low nutritive value

Effects of supplementing willow stem cuttings to ewes grazing drought pastures upon plasma amino acid (AA) concentrations was studied on Massey University's Riverside Farm, near Masteron, on the East Coast of New Zealand. Ewes of similar age and weight (i.e., 59.0 ± 2.22 kg) were assigned to two groups of 7 each, either with (supplemented) or without (control) supplementation of willow, and experimental grazing was carried for 10 weeks from early February until mid April of 2005. Live weight (LW) was recorded fortnightly and body condition score (BCS) was monthly. Blood samples for quantification of plasma amino acids were collected at week 5 and 10. Both groups had a similar pre-grazing pasture mass (i.e., 2000 kg of dry matter/ha) and dead matter content (0.80) with the diet selected by the ewes containing a metabolisable energy (ME) of 8.3 MJ/kg DM, which is typical of drought conditions. The willow was readily eaten, with intake averaging 0.26 kg DM/ewe/d. Willow was of higher ME content than short drought pasture (i.e., 10.1 versus 8.4 MJ/kg DM) and contained condensed tannins at 40.8 ± 1.97 g/kg DM. Both groups of ewes lost live weight at about 50 g/d. Plasma concentration of 3-methyl histidine (88 versus 127 μmol/L) at week 5 and non-essential amino acids (1082 versus 1417 μmol/L) at week 5 and (1155 versus 1324 μmol/L) at week 10, were substantially lower (P<0.05) in willow supplemented versus control ewes, indicating that willow supplementation reduced catabolism of body proteins in ewes under drought feeding conditions.