Fused bicyclic heteroarylpiperazine-substituted l-prolylthiazolidines as highly potent DPP-4 inhibitors lacking the electrophilic nitrile group

Hypoglycemic agents with a mechanism of depeptidyl peptidase IV (DPP-4) inhibition are suitable for once daily oral dosing. It is difficult to strike a balance between inhibitory activity and duration of action in plasma for inhibitors bearing an electrophilic nitrile group. We explored fused bicyclic heteroarylpiperazine substituted at the γ-position of the proline structure in the investigation of l-prolylthiazolidines lacking the electrophilic nitrile. Among them, 2-trifluoroquinolyl compound 8g is the most potent, long-lasting DPP-4 inhibitor (IC(50)=0.37nmol/L) with high selectivity against other related peptidases. X-ray crystal structure determination of 8g indicates that CH-π interactions generated between the quinolyl ring and the guanidinyl group of Arg358 enhances the DPP-4 inhibitory activity and selectivity.     

Discovery and preclinical profile of teneligliptin (3-[(2S,4S)-4-[4-(3-methyl-1-phenyl-1H-pyrazol-5-yl)piperazin-1-yl]pyrrolidin-2-ylcarbonyl]thiazolidine): A highly potent,selective, long-lasting and orally active dipeptidyl peptidase IV inhibitor for the treatment of type 2 diabetes

Dipeptidyl peptidase IV (DPP-4) inhibition is suitable mechanism for once daily oral dosing regimen because of its low risk of hypoglycemia. We explored linked bicyclic heteroarylpiperazines substituted at the γ-position of the proline structure in the course of the investigation of l-prolylthiazolidines. The efforts led to the discovery of a highly potent, selective, long-lasting and orally active DPP-4 inhibitor, 3-[(2S,4S)-4-[4-(3-methyl-1-phenyl-1H-pyrazol-5-yl)piperazin-1-yl]pyrrolidin-2-ylcarbonyl]thiazolidine (8g), which has a unique structure characterized by five consecutive rings. An X-ray co-crystal structure of 8g in DPP-4 demonstrated that the key interaction between the phenyl ring on the pyrazole and the S₂ extensive subsite of DPP-4 not only boosted potency, but also increased selectivity. Compound 8g, at 0.03 mg/kg or higher doses, significantly inhibited the increase of plasma glucose levels after an oral glucose load in Zucker fatty rats. Compound 8g (teneligliptin) has been approved for the treatment of type 2 diabetes in Japan.     

Microalgal cryo-preservation using dimethyl sulfoxide (Me2SO) coupled with two freezing protocols: Influence on the fatty acid profile

Procedures for determining the optimal pre-freezing protocol for cryo-preservation of microalgae are discussed. Three algal species were used (Chlorella vulgaris, Isochrysis galbana and Dunaliella salina) and cryo-stored using two different methods: the slow cooling and the fast freezing. In the slow cooling, each algae batch was treated with or without cryo-protectant (dimethyl sulfoxide: Me₂SO 5% v/v). After 20 min at 4 °C, the midi-straws were filled and cooled slowly (1.5 °C min⁻¹) to −140 °C, by a programmable freezer (Digitcool—IMV), before putting them directly into liquid nitrogen. Fast freezing was performed with 10% or 15% Me₂SO prior to plunging into liquid nitrogen. The three algal species followed the same re-growth pattern as that of the controls. The post-thawed viability with Me₂SO was good for all the selected algae (C. vulgaris >95%, I. galbana and D. salina >70% of the control), applying the slow cooling. The post-thawed viability without Me₂SO was 60% for I. galbana, 52% for D. salina and 33% for C. vulgaris. Fast freezing was not suitable for cryo-storage of I. galbana but gave good post-thawing viability for D. salina (70%). The decrease in fatty acid content of the cryo-stored algae was influenced by the temperature. The rapid decrease in temperature induced by fast freezing can explain the low level of fatty acid content of the three cryo-stored algae. Fatty acid profiles show that the nutritional values of the three cryo-stored micro-algae were not significantly affected especially when treated with slow cooling protocols.     

Interactions d’une restriction calorique avec les effets du Nickel sur la reproduction chez le rat

Human epidemiological studies have demonstrated signs of a reduction, since 1960, of several parameters of the sperm count with an increase of certain male genital tract diseases. The increasing contamination of the environment by chemical compounds appears to be an aetiological factor. Various authors have also proposed the hypothesis that caloric restriction has a beneficial effect on health or longevity. This study was deisgned to compare the effects of nickel on the reproductive functions of rats fed either daily or every second day, in order to evaluate the possible beneficial effects of caloric restriction on rat fertility. This study was conducted with male and female Wistar rats, fed either daily (N), or every second day: intermittent fasting (F). After one month of this treatment, (N) and (F) rats were divided into 2 groups: one group received tap water (NO and FO groups), and the other received the same water enriched with nickel chloride (100 mg/L, NNi and FNi groups). Intermittent fasting was continued in parallel with nickel treatment with for 2, 4, 10, 16, 30 and 60 days. To study malonic dialdehyde (MDA) levels, nickel was administered by intraperitoneal injection at the dosage of 4 mg NiCl₂/kg of body weight for 1, 3, 5 and 10 days. Our results show that nickel induces atrophy of the seminiferous tubules with a reduction of the sperm count and a reduction of serum testosterone levels. A reduction of the number of ovarian follicles was observed in females. Intermittent fasting induced the same types of disturbances with more marked reductions of the number of mobile spermatozoa and serum festosterone levels than those observed after exposure to nickel. The combination of the two factors, fasting and nickel, did not amplify these effects. Analysis of intergroup crosses showed that the pregnancy rate and especially the mean number of implantations were decreased in rats exposed to nickel and/or submitted to intermittent fasting. The lowest pregnancy rate (55%) was observed in (NNi) females crossed with (NO) control males. The smallest number of implantations was observed in (NO) control females crossed with (NNi) males. Nickel did not induce any additional reduction of fertility in rats submitted to intermittent fasting. MDA assays showed that nickel induces lipid peroxidation in ovarian and uterine tissues. However, the relative increase of the MDA level was lower in FNi than NNi rats, i.e. when nickel was associated with intermittent fasting. Our results suggest that nickel and intermittent fasting decrease fertility in rats via two different mechanisms whose effects are not additive. When associated with intermittent fasting, nickel becomes non-toxic, as confirmed by montoring of MDA levels. The low-calorie effect of intermittent fasting could be responsible for inhibition of the cytotoxic effects of metallic nickel classified as an oxidative stress.     

Under low irradiation,the light regime modifies growth and metabolite production in various species of microalgae

The effects of continuous light exposure (24L:0D) and a 12 h:12 h light/dark regime (12L:12D) were compared on the growth and carotenoid, protein, sugar, lipid, and fatty acid contents in Chlorella vulgaris, Nannochloropsis sp., Isochrysis galbana, and Dunaliella salina cultured in a batchwise facility. These microalgae were grown axenically under a low photon flux density (PFD) of 27 μmol photons m^?2 s^?1. C. vulgaris, Nannochloropsis sp., and I. galbana exhibited the highest cell densities when cultured under 24L:0D, whereas D. salina grew better under the alternating light/dark regime. I. galbana accumulated high levels of proteins, sugars, and lipids and exhibited the highest carotenoid content under 24L:0D. Protein production was enhanced in C. vulgaris under 24L:0D. The highest total lipid content was recorded for D. salina, reaching 74.6 % of total proteins, sugars, and lipids in cells at the stationary phase when grown under 12L:12D. The light/dark regime at low PFD was sufficient to stimulate the accumulation of monounsaturated and polyunsaturated fatty acids in all four algae. Their levels, like those of saturated fatty acids, did not differ significantly under the two light regimes. D. salina was an important source of tetradecenoic acid 14:1(n-5). Nannochloropsis sp. produced a large amount of the essential eicosapentaenoic acid, which reached 20 % of total fatty acids under 12L:12D, while I. galbana exhibited the highest level of docosahexaenoic acid, which reached 21 % under both light regimes. This study demonstrated the feasibility of culturing microalgae under low PFD in order to produce large quantities of valuable metabolites, especially various lipids with neutraceutical value.     

Rearing of Fabrea salina Henneguy (Ciliophora, Heterotrichida) with three unicellular feeds

The growth rate of the ciliate Fabrea salina was studied in batch cultures in the presence of three feeds, tested separately from each other: the Prymnesiophyceae, Isochrysis galbana obtained from pure culture, the Chlorophyceae Dunaliella salina, and the commercially available yeast Saccharomyces cerevisiae. F. salina, and D. salina were harvested below the surface from the first evaporation pond and the crystallizer pond, respectively in multi-pond salterns (Sfax, Tunisia). The highest density of Fabrea was recorded with I. galbana (26 ind ml(-1)). However, the greatest length (243 microm) was recorded with Fabrea fed with D. salina. The lowest density, length and biovolume values were recorded with Fabrea fed with S. cerevisiae. The ANOVA test showed that density (F=18, d.f.=57), length (F=33, d.f.=57), and biovolume (F=19, d.f.=57) of Fabrea fed with yeast were significantly different (p<0.001) from those when Fabrea was fed with D. salina and I. galbana. The ciliate Fabrea encountered in the Sfax saltern (Tunisia) might be a valuable food source for Tunisian marine fish hatcheries.     

Discovery and characterization of a new bacterial candidate division by an anaerobic sludge digester metagenomic approach

We have constructed a large fosmid library from a mesophilic anaerobic digester and explored its 16S rDNA diversity using a high-density filter DNA–DNA hybridization procedure. We identified a group of 16S rDNA sequences forming a new bacterial lineage named WWE3 (Waste Water of Evry 3). Only one sequence from the public databases shares a sequence identity above 80% with the WWE3 group which hence cannot be affiliated to any known or candidate prokaryotic division. Despite representing a non-negligible fraction (5% of the 16S rDNA sequences) of the bacterial population of this digester, the WWE3 bacteria could not have been retrieved using the conventional 16S rDNA amplification procedure due to their unusual 16S rDNA gene sequence. WWE3 bacteria were detected by polymerase chain reaction (PCR) in various environments (anaerobic digesters, swine lagoon slurries and freshwater biofilms) using newly designed specific PCR primer sets. Fluorescence in situ hybridization (FISH) analysis of sludge samples showed that WWE3 microorganisms are oval-shaped and located deep inside sludge flocs. Detailed phylogenetic analysis showed that WWE3 bacteria form a distinct monophyletic group deeply branching apart from all known bacterial divisions. A new bacterial candidate division status is proposed for this group.