Optimization of the cryopreservation of African clawed frog (Xenopus laevis) sperm

Cryopreservation of testicular sperm in the African clawed frog, Xenopus laevis, was tested using three penetrating cryoprotectants (DMSO, methanol, and glycerol) and three semen diluents (300 mmol/L glucose, 300 mmol/L sucrose, and a motility inhibiting saline [MIS] solution [150 mmol/L NaCl, 3 mmol/L KCL, 1 mmol/L Mg₂SO₄, 1 mmol/L CaCl₂, and 20 mmol/L Tris, pH 8.0]). Three freezing rates and four thawing rates were also tested, and the best freezing/thawing conditions have been determined. The responses of sperm motility, viability, and fertility were assessed. Incubation of the sperm macerates with penetrating cryoprotectants showed that DMSO was the least toxic and methanol the most toxic. Semen in cryodiluents frozen 10 cm above the surface of liquid nitrogen (freezing rate of 20 to 25 °C/min) and thawed at room temperature for 40 sec had significantly higher percentages of motile and viable sperm than that of semen frozen 5 cm or 8 cm above the surface of liquid nitrogen and thawed at 5, 25, or 30 °C for 10, 15, or 60 sec, respectively. Sperm frozen in MIS containing 5% DMSO had a higher hatching rate than that of sperm frozen in sucrose and glucose diluents containing 5% or 10% DMSO and in MIS containing 10% DMSO. Addition of 73 mmol/L sucrose to the sperm extender MIS + 5% DMSO could improve the postthaw sperm motility and fertility. In conclusion, dilution of collected sperm in MIS solution (to have a final concentration of 6.5 × 10⁶ to 8 × 10⁶/mL) containing 5% DMSO and 73 mmol/L sucrose, freezing in a vapor of liquid nitrogen at 10 cm above the surface, and thawing at room temperature for 40 sec was the best cryopreservation protocol. This protocol gave 70% hatching rate, 80% motility rate, and 75% viability rate of fresh hormonally induced sperm.     

Influence of environmental related concentrations of heavy metals on motility parameters and antioxidant responses in sturgeon sperm

The effects of heavy metals (Cd, Cr and Cd + Cr) on the motility parameters and oxidative stress of sterlet (Acipenser ruthenus) sperm were investigated in vitro. Sturgeon sperm were exposed for 2 h to heavy metals at environmental related concentrations (0.1 mg L^?1 Cr, 0.001 mg L^?1 Cd, 0.1 mg L^?1 Cr + 0.001 mg L^?1 Cd) and higher concentrations (5.0 mg L^?1 Cr, 0.05 mg L^?1 Cd, 5.0 mg L^?1 Cr + 0.05 mg L^?1 Cd). Results revealed that environmental concentrations of heavy metals had no significant influence on motility parameters and antioxidant responses indices in sturgeon sperm, except for LPO level and SOD activity. But higher concentrations of these metals induced oxidative tress in sturgeon sperm in vitro, associated with sperm motility parameters inhibition. Our results suggest that using of sperm in vitro assays may provide a novel and efficiently means for evaluating the effects of residual heavy metals in aquatic environment on sturgeon.     

Expression of ghrelin receptor (GHSR-1a) in rat epididymal spermatozoa and the effects of its activation

In this study we demonstrated the expression of the ghrelin receptor GHSR-1a in rat spermatids and epididymal spermatozoa, as well as some effects of ghrelin on the spermatozoa in vitro. For the demonstration of GHSR-1a the immunocytochemical, immunofluorescence and Western blotting techniques were applied using three different types of antibodies. The response of spermatozoa to ghrelin was tested in a series of in vitro experiments and their effects were evaluated using confocal microscopy and flow cytometry. GHSR-1a protein was found as expressed in the Golgi and acrosomes of spermatids and acrosome regions or the head cell membrane of epididymal spermatozoa. The GHSR-1a expression in spermatozoa was also confirmed by Western blot. No differences were found in percentage of spermatozoa showing annexin-V binding and expression of active form caspase-3 between control and ghrelin-treated spermatozoa. This result may indicate no pro-apoptotic effects of ghrelin neither at 10^?9 nor 10^?6 mol/L concentration. Ghrelin (10^?6 mol/L) increased free intracellular calcium ion concentration in the rat spermatozoa. Moreover, stimulation with 10^?6 mol/L ghrelin increased, while 10^?4 mol/L ghrelin decreased the number of spermatozoa showing progressive motility. In conclusion, the expression of the GHSR-1a receptor in spermatozoa, as well as ghrelin influences on sperm motility and intracellular calcium ion concentration suggest that such biological effects of ghrelin may be produced under in vivo conditions.     

Discovery of highly selective κ-opioid receptor agonists: 10α-Hydroxy TRK-820 derivatives

κ-Opioid receptor agonists with high selectivity over the μ-opioid receptor are attractive targets in the development of drugs for pain and pruritus. We previously reported the synthesis of 10α-hydroxy TRK-820 (1). In this study, we elucidated the biological properties of 1 and optimized its 6-acyl unit by modifying our synthetic route. Among the 10α-hydroxy TRK-820 derivatives prepared, 26 showed the most potent κ-opioid agonist activity (EC₅₀ = 0.00466 nM) and excellent selectivity and 22 was the most κ-selective agonist.     

Cyclic ferrocenylnaphthalene diimide derivative as a new class of G-quadruplex DNA binding ligand

To identify an effective ligand that binds to a G-quadruplex structure but not a double-stranded DNA (dsDNA), a set of biophysical and biochemical experiments were carried out using newly synthesized cyclic ferrocenylnaphthalene diimide (cFNDI, 1) or the non-cyclic derivative (2) with various structures of G-quadruplex DNAs and dsDNA. Compound 1 bound strongly to G-quadruplexes DNAs (10⁶ M^?1 order) with diminished binding to dsDNA (10⁴ M^?1 order) in 100 mM AcOH-AcOK buffer (pH 5.5) containing 100 mM KCl. Interestingly, 1 showed an approximately 50-fold higher selectivity to mixed hybrid-type telomeric G-quadruplex DNA (K = 3.4 × 10⁶ M^?1 and a 2:1 stoichiometry) than dsDNA (K = 7.5 × 10⁴ M^?1) did. Furthermore, 1 showed higher thermal stability to G-quadruplex DNAs than it did to dsDNA with a preference for c-kit and c-myc G-quadruplex DNAs over telomeric and thrombin binding aptamers. Additionally, 1 exhibited telomerase inhibitory activity with a half-maximal inhibitory concentration (IC₅₀) of 0.4 μM. Compound 2 showed a preference for G-quadruplex; however, the binding affinity magnitude and preference were improved in 1 because the former had a cyclic structure.     

Cryopreservation of sperm from seven-band grouper,Epinephelus septemfasciatus

In the present study, we examined methods for the cryopreservation of Epinephelus septemfasciatus spermatozoa. The percent motility, average path velocity, and linearity of movement (LIN) of fresh and corresponding post-thaw sperm were evaluated. Sperm motility was investigated using computer-assisted sperm analysis. Five percent dimethyl sulphoxide (Me₂SO) with 95% fetal bovine serum (FBS) was the most successful cryoprotectant diluent with a comparative post-thaw motility of 77.6 ± 8.5%; 5% dimethyl formamide was also effective. Fetal bovine serum was significantly better as an extender when compared with artificial seminal plasma, glucose, and trehalose solution. Sperm tolerated a wide range of cooling rates (from 27.1 to 94.3 °C min^?1); however, the post-thaw motility of sperm cooled to ?30 °C was significantly lower than that of other cooled temperatures (?40 to ?70 °C). The velocity of post-thaw sperm was significantly lower than that of fresh sperm, although LIN remained the same. For effective cryopreservation of seven-band grouper sperm, samples should be diluted in 5% Me₂SO with 95% FBS and cooled to at least ?40 °C before immersion in liquid nitrogen.     

Influence of organic and inorganic sources of nutrients on the functional diversity of microbial communities in the vegetable cropping system of the Indo-Gangetic plains

The aim of the present study was to assess the effects of different organic and inorganic fertilizers on the functional diversity of soil microbial community under a vegetable production system. The Biolog^® Eco-plate technique and indices, such as average well-colour development (AWCD), McIntosh and Shannon diversity were employed to study the diversity of soil microorganisms. The AWCD, i.e. overall utilization of carbon sources, suggested that different organic treatments had a significant impact on the metabolic activity of soil microorganisms. After 120 h, the highest AWCD values were observed in poultry manure (2.5 t·ha^?1) + vermicompost (3.5 t·ha^?1) (0.63) and farm yard manure (FYM) (10 t·ha^?1) + vermicompost (3.5 t·ha^?1) (0.61). After 72 h, the highest value of the McIntosh diversity index was recorded in poultry manure (2.5 t·ha^?1) + vermicompost (3.5 t·ha^?1) (3.87), followed by poultry manure (2.5 t·ha^?1) + vermicompost (3.5 t·ha^?1) + biofertilizers (Azotobacter 500 g·ha^?1 applied as seed treatment) (3.12). In the case of the Shannon diversity index, the highest values were noticed in organic treatments; however, there was no significant differences between organic and inorganic treatments. Biplot analysis showed a clear differentiation of organic treatments from the inorganic control. The amino acids, phenolics and polymer utilizing microorganisms were dominant in organic treatments. Inorganic control recorded the lowest values of the microbial diversity indices. Through this study, we have identified the best combination of organic nutrients, i.e. poultry manure (2.5 t·ha^?1) + vermicompost (3.5 t·ha^?1) for the stimulation of metabolically active soil microbial communities.     

‘Candidatus Dichloromethanomonas elyunquensis’ gen. nov., sp. nov., a dichloromethane-degrading anaerobe of the Peptococcaceae family

Taxonomic assignments of anaerobic dichloromethane (DCM)-degrading bacteria remain poorly constrained but are important for understanding the microbial diversity of organisms contributing to DCM turnover in environmental systems. We describe the taxonomic classification of a novel DCM degrader in consortium RM obtained from pristine Rio Mameyes sediment. Phylogenetic analysis of full-length 16S rRNA gene sequences demonstrated that the DCM degrader was most closely related to members of the genera Dehalobacter and Syntrophobotulus, but sequence similarities did not exceed 94% and 93%, respectively. Genome-aggregate average amino acid identities against Peptococcaceae members did not exceed 66%, suggesting that the DCM degrader does not affiliate with any described genus. Phylogenetic analysis of conserved single-copy functional genes supported that the DCM degrader represents a novel clade. Growth strictly depended on the presence of DCM, which was consumed at a rate of 160 ± 3 μmol L^?1 d^?1. The DCM degrader attained 5.25 × 10⁷ ± 1.0 × 10⁷ cells per μmol DCM consumed. Fluorescence in situ hybridization revealed rod-shaped cells 4 ± 0.8 μm long and 0.4 ± 0.1 μm wide. Based on the unique phylogenetic, genomic, and physiological characteristics, we propose that the DCM degrader represents a new genus and species, ‘Candidatus Dichloromethanomonas elyunquensis’.     

Effect of entomopathogens on Africanized Apis mellifera L. (Hymenoptera: Apidae)

This study aimed to evaluate the effect of commercially used entomopathogens on Africanized Apis mellifera L. (Hymenoptera: Apidae). Four bioassays were performed: 1) pulverized entomopathogens on A. mellifera; 2) entomopathogens sprayed on a smooth surface; 3) entomopathogens sprayed on soy leaves; and 4) entomopathogens mixed with candy paste (sugar syrup). Five treatments were prepared: sterile distilled water (control), distilled water sterilized with Tween^® 80 (0.01%), and the commercial entomopathogens Metarhizium anisopliae E9 (1.0 × 10⁹ conidia mL^?1), Beauveria bassiana PL63 (1.0 × 10⁸ conidia mL^?1) and Bacillus thuringiensis var. kurstaki HD-1 (3.0 × 10⁸ spores mL^?1). Each treatment consisted of five repetitions, with 20 workers per repetition, which were stored in a plastic box and, later, in a biological oxygen demand (B.O.D.) incubator (27 ± 2 °C, RH of 60% ± 10%, 12-h photophase). The mortality of the workers was evaluated from 1 h to 240 h, and the data were analyzed using Bayesian inference. The workers killed by the ingestion of candy paste contaminated with the pathogens (products) were randomly separated and selected for the removal of the midgut. Each midgut was fixed in Bouin's solution and prepared for histology. B. bassiana was verified to reduce the survival of A. mellifera workers in all bioassays. Moreover, M. anisopliae reduced the survival of A. mellifera workers directly sprayed, on a smooth surface and mixed with candy. B. thuringiensis reduced A. mellifera survival on a smooth surface and mixed with candy paste. However, its effects were lower than that observed by B. bassiana. The treatments with the biological products did not induce morphometric alterations in the midgut of A. mellifera.     

Synthesis and evaluation of the inhibitory activity of the four stereoisomers of the potent and selective human γ-glutamyl transpeptidase inhibitor GGsTop

2-Amino-4-{[3-(carboxymethyl)phenoxy](methoxy)phosphoryl}butanoic acid (GGsTop) is a potent, highly selective, nontoxic, and irreversible inhibitor of γ-glutamyl transpeptidase (GGT). GGsTop has been widely used in academic and medicinal research, and also as an active ingredient (Nahlsgen) in commercial anti-aging cosmetics. GGsTop consists of four stereoisomers due to the presence of two stereogenic centers, i.e., the α-carbon atom of the glutamate mimic (l/d) and the phosphorus atom (R_P/S_P). In this study, each stereoisomer of GGsTop was synthesized stereoselectively and their inhibitory activity against human GGT was evaluated. The l- and d-configurations of each stereoisomer were determined by a combination of a chiral pool synthesis and chiral HPLC analysis. The synthesis of the four stereoisomers of GGsTop used chiral synthetic precursors that were separated by chiral HPLC on a preparative scale. With respect to the configuration of the α-carbon atom of the glutamate mimic, the l-isomer (k_on = 174 M^?1 s^?1) was ca. 8-fold more potent than the d-isomer (k_on = 21.5 M^?1 s^?1). In contrast, the configuration of the phosphorus atom is critical for GGT inhibitory activity. Based on a molecular modeling approach, the absolute configuration of the phosphorus atom of the active GGsTop isomers was postulated to be S_P. The S_P-isomers inhibited human GGT (k_on = 21.5–174 M^?1 s^?1), while the R_P-isomers were inactive even at concentrations of 0.1 mM.     

Kinetics and mathematical modeling of infrared thin-layer drying of garlic slices

Drying of garlic slices in thin-layer have been studied with Infrared (IR) at 0.075, 0.15, 0.225 and 0.3 W cm^?2 radiation intensity and 0.75 and 1.25 m s^?1 air flow velocity. The results showed increasing in drying rate and decreasing at the time of drying with decreasing air flow velocity and increasing IR radiation intensity. The effective moisture diffusivity (D_eff) was obtained using Fick’s diffusion equation and its mean values ranged between 5.83×10^?11 and 7.66×10^?10 m² s^?1 for all investigated conditions. In addition, a third-order polynomial equation linking the effective moisture diffusivity and moisture content was found. Average activation energy increased with the decrease of IR radiation and increase of air flow velocity. Thirteen different mathematical models were verified with non-linear regression analysis for describing the garlic drying process. Modified Henderson and Pabis model presented the best prediction of the drying of garlic slices.     

Bioconversion of phenylpyruvic acid to l-phenylalanine by mixed-gel immobilization of Escherichia coli EP8-10

A mixed-gel of κ-carrageenan and gelatin was used in l-phenylalanine production. The mixed-gel, containing 87.5% κ-carrageenan and 12.5% gelatin [the total gel concentration was 4 wt%], showed the best performance and was selected for further study with Escherichia coli EP8-10. The optimum pH and temperature were 8.5 and 37 °C, respectively. The effects of trehalose and Mg²⁺ were studied in the mixed-gel immobilization. Their optimum concentrations were 5 × 10^?2 and 2 × 10^?3 mol/L, respectively. Under the optimal conditions, 98.3% of the phenylpyruvic acid (PPA) was converted to l-phenylalanine. The activity recovery of the transaminase enzyme in the mixed-gel immobilization was higher than that in single κ-carrageenan immobilization, which was 93.6%. The total PPA conversion rate was over 80% in all 15 batches, suggesting great sustainability in the mixed-gel immobilization. The maximum reaction rate (r_max) was calculated to be 4.75 × 10^?2 mol/(L g h).     

Peripherally acting novel lipo-endomorphin-1 peptides in neuropathic pain without producing constipation

We previously described two novel analogues of endomorphin-1 (Tyr-Pro-Trp-Phe-NH₂, 1), modified with an 8-carbon lipoamino acid (C8LAA) with or without replacement of Tyr¹ with 2,6-dimethyltyrosine (Dmt) at the N-terminus of the peptide (compounds 3 and 4, respectively). They were shown to be more stable and permeable, and acted as potent μ-opioid receptor agonists. In this study we report that the C8LAA modification resulted in successful systemic delivery of both analogues. They produced potent dose-dependent pain relief in a chronic constriction injury-rat model of neuropathic pain after intravenous administration with ED₅₀ values obtained at 6.58 (±1.22) μmol/kg for 3 and 6.18 (±1.17) μmol/kg for 4. Using two different rat models of constipation that assess the effects of μ-opioid receptor agonists on stool hydration and gastro-intestinal motility, compound 3 produced insignificant constipation at 16 μmol/kg, whereas morphine elicited significant constipation at 2 μmol/kg. Compound 3 in contrast to morphine, did not attenuate the hypercapnic ventilatory response at 5 μmol/kg, a dose that fully alleviated hindpaw sensitivity at the time of peak effect in CCI-rats. This finding revealed the lack of respiratory depression effect at antinociceptive dose.     

Freeze-thawing as the factor of spontaneous activation of spermatozoa motility in common carp (Cyprinus carpio L.)

In the present study, we investigated the possibility of spontaneous carp spermatozoa activation by freeze-thawing. To evaluate this, the parameters of spermatozoa motility percentage, velocity, ATP content level and fertility rate of sperm were used. The motility and velocity of spermatozoa activated by freeze-thawing were characterized by motile spermatozoa with a median value of 16% and a velocity of 98 μm/s. In addition, the motility and velocity of sperm from the thawed samples were significantly lower than in the control (median value of 100% for sperm motility and 175 μm/s for sperm velocity). Furthermore, a spontaneously activated spermatozoa motility terminated within five minutes post-thaw time. After freeze-thawing the ATP level significantly decreased with post-thaw time (46 nmol ATP/10⁹ and 10 nmol ATP/10⁹ at 25 s and 10 min after thawing, respectively). Fertility of spermatozoa was not significantly affected within 10 min post-thaw. On the other hand, the fertility of frozen-thawed sperm was significantly lower if compared to fresh sperm. We conclude that the freeze-thawing procedure spontaneously activated spermatozoa motility in common carp. However, this activation did not negatively affect the fertility of frozen-thawed sperm.     

Dopamine D3 receptor antagonists: The quest for a potentially selective PET ligand. Part 3: Radiosynthesis and in vivo studies

Compound 1 is a potent and selective antagonist of the dopamine D₃ receptor. With the aim of developing a carbon-11 labeled ligand for the dopamine D₃ receptor, 1 was selected as a potential PET probe. [¹¹C]1 was obtained by palladium catalyzed cross coupling using [¹¹C]cyanide and 4 with a specific activity of 55.5 ± 25.9 GBq/μmol (1.5 ± 0.7 Ci/μmol). [¹¹C]1 was tested in porcine and non-human primate models to assess its potential as a radioligand for PET imaging of the dopamine D₃ receptor. We conclude that in both species and despite appropriate in vitro properties, [¹¹C]1 does not show any specific signal for the dopamine D₃ receptor.     

Lebetimonas natsushimae sp. nov., a novel strictly anaerobic,moderately thermophilic chemoautotroph isolated from a deep-sea hydrothermal vent polychaete nest in the Mid-Okinawa Trough

A moderately thermophilic, strictly anaerobic, chemoautotrophic bacterium, designated strain HS1857^T, was isolated from a deep-sea hydrothermal vent at the Noho site in the Mid-Okinawa Trough. Strain HS1857^T grew between 35 and 63 °C (optimum 55 °C), in the presence of 10–55 g l^?1 NaCl (optimum 25 g l^?1), and pH 5.5–7.1 (optimum 6.4). Growth occurred with molecular hydrogen as the electron donor and elemental sulfur, nitrate, or selenate as the electron acceptors. Formate could serve as an alternative electron donor with nitrate as an electron acceptor. During growth with nitrate as the electron acceptor, strain HS1857^T produced ammonium and formed a biofilm. CO₂ was utilized as the sole carbon source. The G + C content of the genomic DNA was 33.2 mol%. Phylogenetic analysis of the 16S rRNA gene sequence indicated that strain HS1857^T is a member of the order Nautiliales, showing a sequence similarity of 95.0% with Lebetimonas acidiphila Pd55^T. The fatty acid composition was similar to that of L. acidiphila, which was dominated by C_18:0 (47.0%) and C_18:1 (23.7%). Based on the genomic, chemotaxonomic, phenotypic characteristics, the name Lebetimonas natsushimae sp. nov., is proposed. The type strain is HS1857^T (= NBRC 112478^T = DSM 104102^T).     

Metabolomic markers of fatigue: Association between circulating metabolome and fatigue in women with chronic widespread pain

Background

Fatigue is a sensation of unbearable tiredness that frequently accompanies chronic widespread musculoskeletal pain (CWP) and inflammatory joint disease. Its mechanisms are poorly understood and there is a lack of effective biomarkers for diagnosis and onset prediction. We studied the circulating metabolome in a population sample characterised for CWP to identify biomarkers showing specificity for fatigue.

Antimicrobial activities of Asafoetida and Shirazi thyme essential oils improve the vase life of gerbera cut flowers

This study was conducted to evaluate the chemical composition of asafoetida (Ferula assa-foetida) essential oil (FAEO) and Shirazi thyme (Zataria multiflora) EO (ZMEO) and their impact on vase life of gerbera cut flowers (Gerbera jamesonii cv. Rosalyn). Five concentrations of both, ZMEO and FAEO including 0, 100, 200, 300 and 400 mg L^?1 used as continuous vase solution for gerbera cut flowers. EOs used in this study were extracted by hydrodistillation method using Clevenger apparatus. They were analyzed by GC and GC–MS for determination of the active compounds. Thirty five compounds were identified in ZMEO, mainly including thymol (40.1%), p-cymene (15.5%) and carvacrol (6.5%). Also, thirty compounds were identified in FAEO. The main components were trans propenyl sec-butyl disulfide (21.7%), eudesmol (10-epu-γ) (19.2%) and cis propenyl sec-butyl disulfide (10.2%). The results showed that both ZMEO and FAEO at all concentrations could act as an effective antibacterial compounds and this property increased by increasing their concentration. The results of this research showed that ZMEO increased the vase life at all concentrations but high concentrations of FAEO increased mortality percentage and reduced the vase life of cut flowers. The relative fresh weight and vase solution uptake of gerbera cut flowers increased by the applied EOs treatments. ZMEO at 400 mg L^? 1 and FAEO at 300 and 400 mg L^? 1 resulted the least stem color change. Overall, 200 mg L^? 1 ZMEO and 100 mg L^? 1 FAEO were the best treatments for maintenance of gerbera cut flowers quality during vase life.     

Radiolabeling and initial biological evaluation of [18F]KBM-1 for imaging RAR-α receptors in neuroblastoma

Retinoic acid receptor alpha (RAR-α) plays a significant role in a number of diseases, including neuroblastoma. Children diagnosed with high-risk neuroblastoma are treated 13-cis-retinoic acid, which reduces risk of cancer recurrence. Neuroblastoma cell death is mediated via RAR-α, and expression of RAR-α is upregulated after treatment. A molecular imaging probe that binds RAR-α will help clinicians to diagnose and stratify risk for patients with neuroblastoma, who could benefit from retinoid-based therapy. In this study, we report the radiolabeling, and initial in vivo evaluation of [¹⁸F]KBM-1, a novel RAR-α agonist. The radiochemical synthesis of [¹⁸F]KBM-1 was carried out through KHF₂ assisted substitution of [¹⁸F]^? from aryl-substituted pinacolatoesters-based retinoid precursor. In vitro cell uptake assay in human neuroblastoma cell line showed that the uptake of [¹⁸F]KBM-1 was significantly inhibited by all three blocking agents (KBM-1, ATRA, BD4) at all the selected incubation times. Standard biodistribution in mice bearing neuroblastoma tumors demonstrated increased tumor uptake from 5 min to 60 min post radiotracer injection and the uptake ratios for target to non-target (tumor: muscle) increased 2.2-fold to 3.7-fold from 30 min to 60 min post injection. Tumor uptake in subset of 30 min blocking group was 1.7-fold lower than unblocked. These results demonstrate the potential utility of [¹⁸F]KBM-1 as a RAR-α imaging agent.