Method for hull‐less barley transformation and manipulation of grain mixed‐linkage beta‐glucan

Hull‐less barley is increasingly offering scope for breeding grains with improved characteristics for human nutrition; however, recalcitrance of hull‐less cultivars to transformation has limited the use of these varieties. To overcome this limitation, we sought to develop an effective transformation system for hull‐less barley using the cultivar Torrens. Torrens yielded a transformation efficiency of 1.8%, using a modified Agrobacterium transformation method. This method was used to over‐express genes encoding synthases for the important dietary fiber component, (1,3;1,4)‐β‐glucan (mixed‐linkage glucan), primarily present in starchy endosperm cell walls. Over‐expression of the HvCslF6 gene, driven by an endosperm‐specific promoter, produced lines where mixed‐linkage glucan content increased on average by 45%, peaking at 70% in some lines, with smaller increases in transgenic HvCslH1 grain. Transgenic HvCslF6 lines displayed alterations where grain had a darker color, were more easily crushed than wild type and were smaller. This was associated with an enlarged cavity in the central endosperm and changes in cell morphology, including aleurone and sub‐aleurone cells. This work provides proof‐of‐concept evidence that mixed‐linkage glucan content in hull‐less barley grain can be increased by over‐expression of the HvCslF6 gene, but also indicates that hull‐less cultivars may be more sensitive to attempts to modify cell wall composition.     

Cell-type specific roles for PTEN in establishing a functional retinal architecture

Background

The retina has a unique three-dimensional architecture, the precise organization of which allows for complete sampling of the visual field. Along the radial or apicobasal axis, retinal neurons and their dendritic and axonal arbors are segregated into layers, while perpendicular to this axis, in the tangential plane, four of the six neuronal types form patterned cellular arrays, or mosaics. Currently, the molecular cues that control retinal cell positioning are not well-understood, especially those that operate in the tangential plane. Here we investigated the role of the PTEN phosphatase in establishing a functional retinal architecture.

Methodology/Principal Findings

In the developing retina, PTEN was localized preferentially to ganglion, amacrine and horizontal cells, whose somata are distributed in mosaic patterns in the tangential plane. Generation of a retina-specific Pten knock-out resulted in retinal ganglion, amacrine and horizontal cell hypertrophy, and expansion of the inner plexiform layer. The spacing of Pten mutant mosaic populations was also aberrant, as were the arborization and fasciculation patterns of their processes, displaying cell type-specific defects in the radial and tangential dimensions. Irregular oscillatory potentials were also observed in Pten mutant electroretinograms, indicative of asynchronous amacrine cell firing. Furthermore, while Pten mutant RGC axons targeted appropriate brain regions, optokinetic spatial acuity was reduced in Pten mutant animals. Finally, while some features of the Pten mutant retina appeared similar to those reported in Dscam-mutant mice, PTEN expression and activity were normal in the absence of Dscam.

Conclusions/Significance

We conclude that Pten regulates somal positioning and neurite arborization patterns of a subset of retinal cells that form mosaics, likely functioning independently of Dscam, at least during the embryonic period. Our findings thus reveal an unexpected level of cellular specificity for the multi-purpose phosphatase, and identify Pten as an integral component of a novel cell positioning pathway in the retina.     

HT-29 human colon cancer cell proliferation is regulated by cytosolic phospholipase A2α dependent PGE2via both PKA and PKB pathways

Cytosolic phospholipase A₂α (cPLA₂α) up-regulation has been reported in human colorectal cancer cells, thus we aimed to elucidate its role in the proliferation of the human colorectal cancer cell line, HT-29. EGF caused a rapid activation of cPLA₂α which coincided with a significant increase in cell proliferation. The inhibition of cPLA₂α activity by pyrrophenone or by antisense oligonucleotide against cPLA₂α (AS) or inhibition of prostaglandin E₂ (PGE₂) production by indomethacin resulted with inhibition of cell proliferation, that was restored by addition of PGE₂. The secreted PGE₂ activated both protein kinase A (PKA) and PKB/Akt pathways via the EP2 and EP4 receptors. Either, the PKA inhibitor (H-89) or the PKB/Akt inhibitor (Ly294002) caused a partial inhibition of cell proliferation which was restored by PGE₂. But, inhibited proliferation in the presence of both inhibitors could not be restored by addition of PGE₂. AS or H-89, but not Ly294002, inhibited CREB activation, suggesting that CREB activation is mediated by PKA. AS or Ly294002, but not H-89, decreased PKB/Akt activation as well as the nuclear localization of β-catenin and cyclin D1 and increased the plasma membrane localization of β-catenin with E-cadherin, suggesting that these processes are regulated by the PKB pathway. Similarly, Caco-2 cells exhibited cPLA₂α dependent proliferation via activation of both PKA and PKB/Akt pathways. In conclusion, our findings suggest that the regulation of HT-29 proliferation is mediated by cPLA₂α-dependent PGE₂ production. PGE₂via EP induces CREB phosphorylation by the PKA pathway and regulates β-catenin and cyclin D1 cellular localization by PKB/Akt pathway.     

LOCOMOTOR PERFORMANCE AT LOW TEMPERATURE AND THE EVOLUTION OF NOCTURNALITY IN GECKOS

Nocturnal geckos are active at body temperatures 10–35°C below the thermal optima for maximum rate of aerobic metabolism of diurnal lizards. Therefore, given ancestral (diurnal) lizard physiology, nocturnality causes a substantial thermal handicap in locomotor performance. In prior studies, we hypothesized that a low minimum cost of locomotion (C_min) in geckos was an adaptation that increased locomotor endurance capacity at low, nocturnal temperatures. However, C_min is only part of an integrated system that, in conjunction with the maximum rate of oxygen consumption, sets the maximum speed that can be sustained aerobically (termed the maximum aerobic speed or MAS). We conducted the first phylogenetic analysis of MAS and lizards and found that the greatest changes in MAS, C_min and (at activity temperatures) in the evolutionary history of lizards all coincided with the evolution of nocturnality in geckos. Geckos active at 15–25°C did not become optimized for nocturnal temperatures, or fully offset the thermal effects of nocturnality by evolving maximal rates of oxygen consumption comparable to diurnal lizards active at 35°C. Geckos did evolve MAS twice that of diurnal lizards running at low temperatures by evolving a remarkably low C_min. Allometric analysis and phylogenetically independent contrasts of , C_min, and MAS indicate a 72% evolutionary decrease in , (at activity temperatures) and a 50% evolutionary decrease in C_min concordant with the evolution of nocturnality in geckos. Experimental measurements show that decreased C_min in six species of gecko increased MAS by 50–120% compared to diurnal lizards at low temperatures. Thus, geckos sufficiently overcame the near paralyzing effects of nocturnal temperatures, but only offset about 50% of the decrease in MAS resulting from the low maximum rate of oxygen consumption. Although the nocturnal environment remains severely suboptimal, the evolution of a low cost of locomotion in the ancestor of geckos was highly adaptive for nocturnality. We also present a generalized approach to ecophysiological evolution that integrates phylogeny with the causal relationships among environment, physiology, and performance capacity. With respect to a clade, two hypotheses are central to our integrative approach: (1) a change of an environmental variable (e.g., temperature) causes a performance handicap; and (2) evolution of a physiological variable (e.g., minimum cost of locomotion [C_min]) increases performance in the derived environment. To test the hypothesis that evolution of a physiological variable is adaptive in nature, we suggest determining if individuals in nature perform at levels exceeding the performance capacity of their hypothetical ancestors and if this additional performance capacity is due to the evolution of the physiological variable in question.     

Mannan structural complexity is decreased when Candida albicans is cultivated in blood or serum at physiological temperature

The Candida albicans cell wall provides an architecture that allows for the organism to survive environmental stress as well as interaction with host tissues. Previous work has focused on growing C. albicans on media such as Sabouraud or YPD at 30 °C. Because C. albicans normally colonizes a host, we hypothesized that cultivation on blood or serum at 37 °C would result in structural changes in cell wall mannan. C. albicans SC5314 was inoculated onto YPD, 5% blood, or 5% serum agar media three successive times at 30 °C and 37 °C, then cultivated overnight at 30 °C in YPD. The mannan was extracted and characterized using 1D and 2D ¹H NMR techniques. At 30 °C cells grown in blood and serum contain less acid-stable terminal β-(1→2)-linked d-mannose and α-(1→2)-linked d-mannose-containing side chains, while the acid-labile side chains of mannan grown in blood and serum contain fewer β-Man-(1→2)-α-Man-(1→ side chains. The decrement in acid-stable mannan side chains is greater at 37 °C than at 30 °C. Cells grown on blood at 37 °C show fewer →6)-α-Man-(1→ structural motifs in the acid-stable polymer backbone. The data indicate that C. albicans, grown on media containing host-derived components, produces less complex mannan. This is accentuated when the cells are cultured at 37 °C. This study demonstrates that the C. albicans cell wall is a dynamic and adaptive organelle, which alters its structural phenotype in response to growth in host-derived media at physiological temperature.     

Effect of acetylcholinesterase inhibition with pyridostigmine on cardiac parasympathetic function in sedentary adults and trained athletes

Heart rate variability and postexercise heart rate recovery are used to assess cardiac parasympathetic tone in human studies, but in some cases these indexes appear to yield discordant information. We utilized pyridostigmine, an acetylcholinesterase inhibitor that selectively augments the parasympathetic efferent signal, to further characterize parasympathetic regulation of rest and postexercise heart rate. We measured time- and frequency-domain indexes of resting heart rate variability and postexercise heart rate recovery in 10 sedentary adults and 10 aerobically trained athletes after a single oral dose of pyridostigmine (30 mg) and matching placebo in randomized, double-blind, crossover trial. In sedentary adults, pyridostigmine decreased resting heart rate [from 66.7 (SD 12.6) to 58.1 beats/min (SD 7.6), P = 0.005 vs. placebo] and increased postexercise heart rate recovery at 1 min [from 40.7 (SD 10.9) to 45.1 beats/min (SD 8.8), P = 0.02 vs. placebo]. In trained athletes, pyridostigmine did not change resting heart rate or postexercise heart rate recovery when compared with placebo. Time- and frequency-domain indexes of resting heart rate variability did not differ after pyridostigmine versus placebo in either cohort and were not significantly associated with postexercise heart rate recovery in either cohort. The divergent effects of pyridostigmine on resting and postexercise measures of cardiac parasympathetic function in sedentary subjects confirm that these measures characterize distinct aspects of cardiac parasympathetic regulation. The lesser effect of pyridostigmine on either measure of cardiac parasympathetic tone in the trained athletes indicates that the enhanced parasympathetic tone associated with exercise training is at least partially attributable to adaptations in the efferent parasympathetic pathway.     

Molting-specific downregulation of C. elegans body-wall muscle attachment sites: The role of RNF-5 E3 ligase

Repeated molting of the cuticula is an integral part of arthropod and nematode development. Shedding of the old cuticle takes place on the surface of hypodermal cells, which are also responsible for secretion and synthesis of a new cuticle. Here, we use the model nematode Caenorhabditis elegans to show that muscle cells, laying beneath and mechanically linked to the hypodermis, play an important role during molting. We followed the molecular composition and distribution of integrin mediated adhesion structures called dense bodies (DB), which indirectly connect muscles to the hypodermis. We found the concentration of two DB proteins (PAT-3/β-integrin and UNC-95) to decrease during the quiescent phase of molting, concomitant with an apparent increase in lateral movement of the DB. We show that levels of the E3-ligase RNF-5 increase specifically during molting, and that RNF-5 acts to ubiquitinate the DB protein UNC-95. Persistent high levels of RNF-5 driven by a heatshock or unc-95 promoter lead to failure of ecdysis, and in non-molting worms to a progressive detachment of the cuticle from the hypodermis. These observations indicate that increased DB dynamics characterizes the lethargus phase of molting in parallel to decreased levels of DB components and that temporal expression of RNF-5 contributes to an efficient molting process.