Glycosylation of inositol phosphorylceramide sphingolipids is required for normal growth and reproduction in Arabidopsis

Sphingolipids are a major component of plant plasma membranes and endomembranes, and mediate a diverse range of biological processes. Study of the highly glycosylated glycosyl inositol phosphorylceramide (GIPC) sphingolipids has been slow as a result of challenges associated with the extractability of GIPCs, and their functions in the plant remain poorly characterized. We recently discovered an Arabidopsis GIPC glucuronosyltransferase, INOSITOL PHOSPHORYLCERAMIDE GLUCURONOSYLTRANSFERASE 1 (IPUT1), which is the first enzyme in the GIPC glycosylation pathway. Plants homozygous for the iput1 loss‐of‐function mutation were unobtainable, and so the developmental effects of reduced GIPC glucuronosylation could not be analyzed in planta. Using a pollen‐specific rescue construct, we have here isolated homozygous iput1 mutants. The iput1 mutants show severe dwarfism, compromised pollen tube guidance, and constitutive activation of salicyclic acid‐mediated defense pathways. The mutants also possess reduced GIPCs, increased ceramides, and an increased incorporation of short‐chain fatty acids and dihydroxylated bases into inositol phosphorylceramides and GIPCs. The assignment of a direct role for GIPC glycan head groups in the impaired processes in iput1 mutants is complicated by the vast compensatory changes in the sphingolipidome; however, our results reveal that the glycosylation steps of GIPC biosynthesis are important regulated components of sphingolipid metabolism. This study corroborates previously suggested roles for GIPC glycans in plant growth and defense, suggests important roles for them in reproduction and demonstrates that the entire sphingolipidome is sensitive to their status.     

Natural abenquines and synthetic analogues: Preliminary exploration of their cytotoxic activity

In this study, we explore the cytotoxic activity of four natural abenquines (2a–d) and fourteen synthetic analogues (2e–j and 3a–h) against a panel of six human cancer cell lines using a SRB assay. It was found that most of the compounds revealed higher levels of cytotoxic activities than naturally occurring abenquines. The analogues carrying ethylpyrrolidinyl and ethylpyrimidinyl with either an acetyl group (2 h–i) or a benzoyl group (3f–g), were the most potent against all human cancer cell lines and displayed EC₅₀ between a range of 0.6–3.4 μM. Notably, of the compounds tested, compound 2i proved the most cytotoxic against both ovarian (A2780) and breast (MCF7) cells, showing EC₅₀ = 0.6 and 0.8 μM respectively. Likewise, the analogues 2i, 3f and 3 g showed strong activity against cell HT29 with EC₅₀ = 0.9 μM for these compounds.     

A multiplexed magnetic tweezer with precision particle tracking and bi-directional force control

Background

In the past two decades, methods have been developed to measure the mechanical properties of single biomolecules. One of these methods, Magnetic tweezers, is amenable to acquisition of data on many single molecules simultaneously, but to take full advantage of this "multiplexing" ability, it is necessary to simultaneously incorporate many capabilities that have been only demonstrated separately.

Methods

Our custom built magnetic tweezer combines high multiplexing, precision bead tracking, and bi-directional force control into a flexible and stable platform for examining single molecule behavior. This was accomplished using electromagnets, which provide high temporal control of force while achieving force levels similar to permanent magnets via large paramagnetic beads.

Results

Here we describe the instrument and its ability to apply 2–260 pN of force on up to 120 beads simultaneously, with a maximum spatial precision of 12 nm using a variety of bead sizes and experimental techniques. We also demonstrate a novel method for increasing the precision of force estimations on heterogeneous paramagnetic beads using a combination of density separation and bi-directional force correlation which reduces the coefficient of variation of force from 27% to 6%. We then use the instrument to examine the force dependence of uncoiling and recoiling velocity of type 1 fimbriae from Eschericia coli (E. coli) bacteria, and see similar results to previous studies.

Conclusion

This platform provides a simple, effective, and flexible method for efficiently gathering single molecule force spectroscopy measurements.

     

An automatic device for in vivo absorption spectra acquisition and chlorophyll estimation in phytoplankton cultures

In order to aid the study of photoacclimation, a new programmable deviceis described which provides automatic on-line acquisition of in vivo cellabsorption in phytoplankton cultures. The system was used for a long-termstudy of Rhodomonas salina grown at constant photon flux density ina nitrate-limited continuous culture with different dilution rates. Particulate absorption measured at the red chlorophyll a (Chl a)maximum was not a good proxy of biomass, because of the large variabilityof cellular chlorophyll induced by nitrogen limitation. However, thedevice is well suited to automatic assessment of Chl a andphycoerythrin (PE) concentrations in phytoplankton cultures, if algal cellsize and concentration are measured in parallel to correct the packagingeffect. The effects of nitrogen limitation on Chl a and PE contentsand particle absorbance are discussed.     

Effects of Rice Seed Surface Sterilization with Hypochlorite on Inoculated Burkholderia vietnamiensis

When a combination of hydrogen peroxide and hypochlorite was used to surface sterilize rice seeds, a 10²- to 10⁴-fold decrease in CFU was observed during the first 15 h after inoculation of the rice rhizosphere organism Burkholderia vietnamiensis TVV75. This artifact could not be eliminated simply by rinsing the seeds, even thoroughly, with sterile distilled water. When growth resumed, a significant increase in the frequency of rifampin- and nalidixic acid-resistant mutants in the population was observed compared to the control without seeds. This phenomenon was a specific effect of hypochlorite; it was not observed with hydrogen peroxide alone. It was also not observed when the effect of hypochlorite was counteracted by sodium thiosulfate. We hypothesized that the hypochlorite used for disinfection reacted with the rice seed surface, forming a chlorine cover which was not removed by rinsing and generated mutagenic chloramines. We studied a set of rifampin- and nalidixic acid-resistant mutants obtained after seed surface sterilization. The corresponding rpoB and gyrA genes were amplified and sequenced to characterize the induced mutations. The mutations in five of seven nalidixic acid-resistant mutants and all of the rifampin-resistant mutants studied were found to correspond to single amino acid substitutions. Hypochlorite surface sterilization can thus be a source of artifacts when the initial bacterial colonization of a plant is studied.     

2-Cyano-3,10-dioxooleana-1,9(11)-dien-28-oic acid anhydride. A novel and highly potent anti-inflammatory and cytoprotective agent

2-Cyano-3,10-dioxooleana-1,9(11)-dien-28-oic acid anhydride (CDDO anhydride) has been synthesized, which is the first example of an oleanane triterpenoid anhydride. CDDO anhydride shows potency similar to or higher than the corresponding acid (CDDO) in various in vitro and in vivo assays related to inflammation and carcinogenesis. Notably, preliminary phamacokinetics studies show that CDDO anhydride levels are higher than CDDO levels in mouse tissues and blood. Further evaluation of CDDO anhydride is in progress.     

Aggressive behaviour in subadult and adult black redstarts: status-signalling strategy?

In some birds, yearling males (subadults) are less brightly coloured than the adult ones. To explain this phenomenon known as delayed plumage maturation (DPM), several hypotheses have recently been proposed. The aim of this paper was to test predictions of status signal hypothesis (SSH). This hypothesis claims that the adults less frequently attack dull coloured subadult males, and consequently DPM facilitates survival of subadults during the first breeding season. The experiments were performed in two habitats in the Prague suburbs: a housing estate and garden city. Subadult and adult males of the black redstart (Phoenicurus ochruros) were exposed to stuffed dummies of both age categories. Contrary to predictions of SSH, brown (subadult like) coloration of the dummy had no suppressive effect on defensive behaviour of the resident males. On the other hand, subadult males were more active in vocal territorial defence such as singing and producing snarl and whistle sounds during the experiments. The defenders of smaller territories generally behaved more aggressively than defenders of large territories in habitat of garden city, and especially adult males responded more aggressively against dummies in this habitat. In a housing estate, a habitat of evenly distributed resources (i.e. vertical surfaces), the birds tended to observe the dummy prior to attack it directly.     

Chronological vs. Physiological Age as Determinants of Mating Decisions: Studies on Female Choice Over Lifespan in An Acoustic Moth

Life history theory predicts that females may adjust the selectivity expressed in mate choice as they age. Particularly in cases where time is limiting, females are expected to reduce selectivity and thereby avoid losing a terminal mating opportunity. Some evidence for this reduction has been found in vertebrates and long‐lived insects, and several recent findings show that it may also exist in insects with very short adult longevities. Theory also predicts that behavioural adjustments should respond to remaining longevity (physiological age) rather than chronological age, but very little information relevant to this issue exists. We studied age‐related changes in mating behaviour in an acoustic moth species (Achroia grisella; Lepidoptera: Pyralidae) in which females choose males based on the intensity, rhythm and other temporal features of the male song, with the objective of determining whether observed adjustments reflect chronological or physiological age. In accordance with theory, we found that females became less selective in their evaluation of male song rhythm as they aged and that this adjustment was predicted by physiological rather than chronological age. The reduction in selectivity may be due to decreased movement and searching, factors that can prevent a female from perceiving all of the male songs being broadcast locally in a complex environment where the transmission of some songs is blocked in some locations. We did not observe these age‐related effects in a simpler, open environment.     

In vitro strategies for human gametes production from stem cells

Embryonic stem cells (ESC) are self-renewal and pluripotent cells that are able to differentiate in vitro into several cell types in favourable conditions. Technical protocols for in vitro gametes production have been developed in mice and human species. The functionality of such differentiated cells is not always analysed and an early meiotic arrest is a current observation. These kinds of experimentations have also been tested from human induced pluripotent stem cells (IPSC). However, differentiation ends shortly at the primordial germ cell stage.