High resolution ultrasound-guided microinjection for interventional studies of early embryonic and placental development in vivo in mice

Background  

In utero microinjection has proven valuable for exploring the developmental consequences of altering gene expression, and for studying cell lineage or migration during the latter half of embryonic mouse development (from embryonic day 9.5 of gestation (E9.5)). In the current study, we use ultrasound guidance to accurately target microinjections in the conceptus at E6.5–E7.5, which is prior to cardiovascular or placental dependence. This method may be useful for determining the developmental effects of targeted genetic or cellular interventions at critical stages of placentation, gastrulation, axis formation, and neural tube closure.     

Inhibitor studies on non-photochemical plastoquinone reduction and H2 photoproduction in Chlamydomonas reinhardtii

In the absence of PSII, non-photochemical reduction of plastoquinones (PQs) occurs following NADH or NADPH addition in thylakoid membranes of the green alga Chlamydomonas reinhardtii. The nature of the enzyme involved in this reaction has been investigated in vitro by measuring chlorophyll fluorescence increase in anoxia and light-dependent O₂ uptake in the presence of methyl viologen. Based on the insensitivity of these reactions to rotenone, a type-I NADH dehydrogenase (NDH-1) inhibitor, and their sensitivity to flavoenzyme inhibitors and thiol blocking agents, we conclude to the involvement of a type-II NADH dehydrogenase (NDH-2) in PQ reduction. Intact Chlamydomonas cells placed in anoxia have the property to produce H₂ in the light by a Fe-hydrogenase which uses reduced ferredoxin as an electron donor. H₂ production also occurs in the absence of PSII thanks to the existence of a non-photochemical pathway of PQ reduction. From inhibitors effects, we suggest the involvement of a plastidial NDH-2 in PSII-independent H₂ production in Chlamydomonas. These results are discussed in relation to the absence of ndh genes in Chlamydomonas plastid genome and to the existence of 7 ORFs homologous to type-II NDHs in its nuclear genome.     

Induction of N-acetyllactosamine synthetase activity in the mouse mammary gland by prolactin and placental lactogen hormone]

The authors show that the ovine prolactine promote induction of N. acetyl lactosamine synthetase in tissue culture of mammary glands of pregnant mice. A crude extract of human placenta has also a lactogenic activity as tested by the same method, but in this case the blank values are very high for large concentration of crude extract. The molecular forms of HCS are tested: the slow band has a lactogenic activity, the intermediate band has no activity and the rapid band seems to be inhibitory.     

Autotrophic and mixotrophic hydrogen photoproduction in sulfur-deprived chlamydomonas cells

In Chlamydomonas reinhardtii cells, H2 photoproduction can be induced in conditions of sulfur deprivation in the presence of acetate. The decrease in photosystem II (PSII) activity induced by sulfur deprivation leads to anoxia, respiration becoming higher than photosynthesis, thereby allowing H2 production. Two different electron transfer pathways, one PSII dependent and the other PSII independent, have been proposed to account for H2 photoproduction. In this study, we investigated the contribution of both pathways as well as the acetate requirement for H2 production in conditions of sulfur deficiency. By using 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), a PSII inhibitor, which was added at different times after the beginning of sulfur deprivation, we show that PSII-independent H2 photoproduction depends on previously accumulated starch resulting from previous photosynthetic activity. Starch accumulation was observed in response to sulfur deprivation in mixotrophic conditions (presence of acetate) but also in photoautotrophic conditions. However, no H2 production was measured in photoautotrophy if PSII was not inhibited by DCMU, due to the fact that anoxia was not reached. When DCMU was added at optimal starch accumulation, significant H2 production was measured. H2 production was enhanced in autotrophic conditions by removing O2 using N2 bubbling, thereby showing that substantial H2 production can be achieved in the absence of acetate by using the PSII-independent pathway. Based on these data, we discuss the possibilities of designing autotrophic protocols for algal H2 photoproduction.     

Impact of Ramadan intermittent fasting on cognitive function in trained cyclists: a pilot study

This study assessed selected measures of cognitive function in trained cyclists who observed daylight fasting during Ramadan. Eleven cyclists volunteered to participate (age: 21.6±4.8 years, VO₂max: 57.7±5.6 ml kg⁻¹·min⁻¹) and were followed for 2 months. Cognitive function (Cambridge Neuropsychological Test Automated Battery (CANTAB), Reaction Time index (RTI) and Rapid Visual Information Processing (RVP) tests) and sleep architecture (ambulatory EEG) were assessed: before Ramadan (BR), in the 1st week (RA1) and 4th week of Ramadan (RA4), and 2 weeks post-Ramadan (PR). Both cognitive tests were performed twice per day: before and after Ramadan at 8-10 a.m. and 4-6 p.m., and during Ramadan at 4-6 p.m. and 0-2 a.m., respectively. Training load (TL) by the rating of perceived exertion (RPE) method and wellness (Hooper index) were measured daily. If the TL increased over the study period, this variable was stable during Ramadan. The perceived fatigue and delayed onset muscle soreness (DOMS) increased at RA4. Sleep patterns and architecture showed clear disturbances, with significant increases in the number of awakenings and light sleep durations during Ramadan (RA1 and RA4), together with decreased durations of deep and REM sleep stages at PR. RTI (simple and multiple reaction index) reaction and movement times did not vary over the study period. The RVP test showed reduced false alarms during Ramadan, suggesting reduced impulsivity. Overall accuracy significantly increased at RA1, RA4 and PR compared to baseline. At RA4, the accuracy was higher at 0-2 a.m. compared to 4-6 p.m. Despite the observed disturbances in sleep architecture, Ramadan fasting did not negatively impact the cognitive performance of trained cyclists from the Middle East.     

Description and analysis of genetic diversity between commercial bean lines (Phaseolus vulgaris L.)

The effectiveness of RFLP, DAMD-PCR, ISSR and RAPD markers in assessing polymorphism and relationships between 24 commercial lines of Phaseolus vulgaris L.was evaluated. We have used a Phaseolus-specific minisatellite sequence as a probe, which enabled 23 of the bean lines tested to be fingerprinted. Based on the sequence information obtained, primers corresponding to the bean-specific minisatellite core sequence were used in subsequent PCR amplifications. Our observations indicated that while the DAMD-PCR was sensitive in detecting genetic variation between bean species and between accessions of P. vulgaris, when used alone it may be limited in its ability to detect genetic variation among cultivated bean lines due to the low number of loci amplified. Only one out of the five ISSR primers tested was efficient in generating multiple band profiles, which was insufficient to distinguish all the different bean lines. Reproducible RAPD profiles were obtained, and these allowed us to differentiate all the genotypes tested with seven primers. We ultimately used only results from RFLP and RAPD markers to explore the genetic diversity among commercial bean lines. Both analyses led to the same clustering of the bean lines according to their geographical origins (United States or Europe). With respect to the European lines, the results obtained from RAPD data also enable the lines to be clustered according to their creators. Received: 15 January 2000 / Accepted: 21 March 2000     

Effect of beta-carotene administration on reproductive function of horse and pony mares

The objective of this study was to determine whether supplemental beta-carotene would influence reproductive function in mares maintained on spring and summer pastures and to characterize plasma carotene concentrations during the estrous cycle. Carotene concentrations in plasma did not vary with day of estrous cycle (P = 0.7455). Mares receiving every other day injections of beta-carotene (400 mg; n = 4) or saline (10 ml; n = 4) during proestrus/estrus did not differ in plasma estradiol (E(2)) concentrations (P = 0.6313), follicle development (P = 0.8068), or plasma progesterone (P(4)) concentrations during the following diestrus (P = 0.4954). Moreover, no differences in plasma P(4) concentrations (P = 0.9047) were detected between mares receiving every other day injections of beta-carotene (400 mg; n = 4) or saline (10 ml; n = 4) during diestrus. However, administration of beta-carotene raised plasma carotene concentrations relative to controls when injected during proestrus/estrus (P = 0.0096) and diestrus (P = 0.0099). Pregnancy rates (P = 0.4900) and number of cycles required for pregnancy (P = 0.2880) were similar for mares administered injections of saline (10 ml; n = 37), beta-carotene (400 mg; n = 37), vitamin A (160,000 IU; n = 38), or vitamin A + beta-carotene (160,000 IU + 400 mg; n = 43), on the first or second day of estrus and on the day of breeding. Therefore, these results collectively suggest that supplemental beta-carotene does not affect the reproductive function of mares fed adequate dietary carotene. Whether supplemental beta-carotene would enhance reproductive function in mares on low carotene diets warrants further investigation.     

Microalgae Synthesize Hydrocarbons from Long-Chain Fatty Acids via a Light-Dependent Pathway

Microalgae are considered a promising platform for the production of lipid-based biofuels. While oil accumulation pathways are intensively researched, the possible existence of a microalgal pathways converting fatty acids into alka(e)nes has received little attention. Here, we provide evidence that such a pathway occurs in several microalgal species from the green and the red lineages. In Chlamydomonas reinhardtii (Chlorophyceae), a C17 alkene, n-heptadecene, was detected in the cell pellet and the headspace of liquid cultures. The Chlamydomonas alkene was identified as 7-heptadecene, an isomer likely formed by decarboxylation of cis-vaccenic acid. Accordingly, incubation of intact Chlamydomonas cells with per-deuterated D₃₁-16:0 (palmitic) acid yielded D₃₁-18:0 (stearic) acid, D₂₉-18:1 (oleic and cis-vaccenic) acids, and D₂₉-heptadecene. These findings showed that loss of the carboxyl group of a C₁₈ monounsaturated fatty acid lead to heptadecene formation. Amount of 7-heptadecene varied with growth phase and temperature and was strictly dependent on light but was not affected by an inhibitor of photosystem II. Cell fractionation showed that approximately 80% of the alkene is localized in the chloroplast. Heptadecane, pentadecane, as well as 7- and 8-heptadecene were detected in Chlorella variabilis NC64A (Trebouxiophyceae) and several Nannochloropsis species (Eustigmatophyceae). In contrast, Ostreococcus tauri (Mamiellophyceae) and the diatom Phaeodactylum tricornutum produced C₂₁ hexaene, without detectable C₁₅-C₁₉ hydrocarbons. Interestingly, no homologs of known hydrocarbon biosynthesis genes were found in the Nannochloropsis, Chlorella, or Chlamydomonas genomes. This work thus demonstrates that microalgae have the ability to convert C₁₆ and C₁₈ fatty acids into alka(e)nes by a new, light-dependent pathway.Hydrocarbons derived from fatty acids (i.e. alkanes and alkenes) are ubiquitous in plant and insect outermost tissues where they often represent a major part of cuticular waxes and play an essential role in preventing water loss from the organisms to the dry terrestrial environment (Hadley, 1989; Kunst et al., 2005). In several insect species, select cuticular alkenes also act as sex pheromones (Wicker-Thomas and Chertemps, 2010). Occurrence of alkanes or alkenes has also been reported in various microorganisms (Ladygina et al., 2006; Wang and Lu, 2013). For example, synthesis of hydrocarbons is widespread in cyanobacteria (Coates et al., 2014), and it is thought that cyanobacterial alka(e)nes contribute significantly to the hydrocarbon cycle of the upper ocean (Lea-Smith et al., 2015).Alka(e)nes of various chain lengths are important targets for biotechnology because they are major components of gasoline (mainly C5-C9 hydrocarbons), jet fuels (C5-C16), and diesel fuels (C12-C20). The alkane biosynthetic pathway of plants has been partly elucidated (Lee and Suh, 2013; Bernard and Joubès, 2013), but the use of plant hydrocarbons as a renewable source of liquid fuels is hampered by predominance of constituents with high carbon numbers (>C25), which entails solid state at ambient temperature (Jetter and Kunst, 2008). Therefore, there is great interest in the microbial pathways of hydrocarbon synthesis producing shorter chain compounds (C15-C19). In cyanobacteria, hydrocarbons are produced by two distinct pathways. The first one comprises the sequential action of an acyl-ACP reductase transforming a C15-C19 fatty acyl-ACP into a fatty aldehyde and an aldehyde-deformylating oxygenase catalyzing the oxidative cleavage of the fatty aldehyde into alka(e)ne and formic acid (Schirmer et al., 2010; Li et al., 2012). The second pathway involves a type I polyketide synthase that elongates and decarboxylates fatty acids to form alkenes with a terminal double bond (Mendez-Perez et al., 2011). Additional pathways of alkene synthesis have been described in bacteria. In Micrococcus luteus, a three-gene cluster has been shown to control the head-to-head condensation of fatty acids to form very-long-chain alkenes with internal double bonds (Beller et al., 2010). Direct decarboxylation of a long-chain fatty acid into a terminal alkene has also been reported and is catalyzed by a cytochrome P450 in Jeotgalicoccus spp. (Rude et al., 2011) and by a nonheme iron oxidase in Pseudomonas (Rui et al., 2014).Among microbes, microalgae would be ideally suited to harness the synthesis of hydrocarbons from fatty acid precursors because they are photosynthetic organisms combining a high biomass productivity (León-Bañares et al., 2004; Beer et al., 2009; Malcata, 2011; Wijffels et al., 2013) and a strong capacity to synthesize and accumulate fatty acids (Hu et al., 2008; Harwood and Guschina, 2009; Liu and Benning 2013). However, studies on microalgal alka(e)ne synthesis are scarce. In some diatoms and other algal species, a very-long-chain alkene, a C21 hexaene, has been found (Lee et al., 1970; Lee and Loeblich, 1971). Other very-long-chain alkenes have been described in the slow-growing colonial Chlorophycea Botryococcus brauni, which excretes a variety of hydrophobic compounds including C27 n-alkadienes (Metzger and Largeau, 2005; Jin et al., 2016). A decarbonylase activity converting a fatty n-aldehyde substrate to a n-alkane has been shown in B. brauni (Dennis and Kolattukudy, 1992); however, the corresponding protein has not been identified so far. Presence of shorter chain alka(e)nes in some microalga species has been reported in the context of geochemical studies (Han et al., 1968; Gelpi et al., 1970; Tornabene et al., 1980; Afi et al., 1996) but the biology of these compounds has not been investigated further.Here, we show that alka(e)nes with C15 to C17 chains can be detected in several model microalgae and originate from fatty acid metabolism. In Chlamydomonas reinhardtii and Chlorella variabilis NC64A, 7-heptadecene is identified as the major hydrocarbon produced, and we demonstrate that its synthesis depends strictly on light and uses cis-vaccenic acid as a precursor. We also show the presence of C15 to C17 alka(e)nes in Nannochloropsis sp., a model microalga from the red lineage. Absence of homologs to known hydrocarbon synthesis genes in the genomes of Chlamydomonas, Chlorella, and Nannochloropsis indicates that a hitherto unknown type of alka(e)ne-producing pathway operates in these microalgae. The wide occurrence of microalgae in marine environments suggests that microalgal alka(e)nes contribute significantly to the ocean’s hydrocarbon cycle.