Multimers of anionic amphiphiles mimic calmodulin stimulation of cyclic nucleotide phosphodiesterase

Oleic acid, phosphatidylserine and pyrenedecanoic acid were found to activate calmodulin-deficient cyclic nucleotide phosphodiesterase at concentrations above their critical micellar concentration. In contrast with calmodulin these activators do not require the presence of Ca2+ for their action. It is shown that the size of phosphatidylserine vesicles is of crucial importance with respect to the activating potency of phosphatidylserine. Fluorescence measurements with the probe pyrenedecanoic acid revealed that micelles rather than monomers are the active species for stimulation of phosphodiesterase. There are indications that this result also may be applied to the other activators.     

Electron Microscopic Study of Development in a Sea Cucumber,Stichopus tremulus (Holothuroidea), from Unfertilized Egg through Hatched Blastula

In this, the first fine structural study of sea cucumber embryology, eggs and embryos of Stichopus tremulus developing at 7.5°C are described from spawning through hatched blastulae. Spawned eggs are at about first meiotic metaphase and are surrounded by a jelly layer that remains around the embryos until hatching. No vitelline coat can be demonstrated, but whether it is truly absent or removed by electron microscopic processing is not known. Insemination initiates a rapid cortical reaction, completed within 2 min., which involves a wave of cortical granule exocytosis and fertilization envelope formation. The compactly fibrous fertilization envelope is about 50 nm thick and appears to consist entirely of ejected cortical granule material (if one assumes that there is no vitelline coat). As the fertilization envelope elevates, no hyaline layer appears in the perivitelline space. The first and second polar bodies are emitted, respectively, at about 9 and 15 min. after insemination. The first seven or so cleavages are equal, radial, and occur approximately every 4 hr. The blastocoel opens up at the four-cell stage and, during the earlier cleavages, remains connected with the perivitelline space via numerous gaps between the roughly spherical blastomeres. At the 64-cell stage, these gaps begin to close as the blastomeres start to become cuboidal; in addition, an embryonic cuticle is produced on the apical surface of each blastomere. In embryos of several hundred cells, the blastomeres become associated apicolaterally by junctional complexes, each consisting of a zonula adherens and a septate junction. Several hours before hatching, a single cilium is produced at the apical surface of most blastomeres. At hatching (about 50 hr after insemination), the ciliated blastula leaves behind the fertilization envelope and jelly layer. Swimming blastulae soon begin to elongate in the animal-vegetal axis, and a basal lamina develops on blastomere surfaces facing the blastocoel. The discussion includes a fine structural comparison of egg coats among the five classes of the phylum Echinodermata.     

Differentiation between acetate and higher volatile acids in the modeling of the anaerobic biomethanation process

This paper presents a model for the single-stage completely-mixed anaerobic digestion of complex substrates containing no volatile acids. In the model, volatile acids produced by the acidogenic bacteria are no longer considered together. Acetate is assumed to be representative of the substrate and propionate and butyrate act only as inhibitors for the methanogenic bacteria.Nomenclature ···0 represents factors associated with the influent - ···1 represents factors associated with the acidogenic bacteria - ···2 represents factors associated with the methanogenic bacteria - Q hydraulic flow (1/d) - V reactor liquid volume (1) - T temperature of the mixed liquor (° C) - S microorganisms concentration (mg/1) - L volatile solids concentration (mg VS/1) - Lb biodegradable volatile solids concentration (mg VS/1) - VA2 acetate concentration (mg/1) - VA3 volatile acids with 3 to 5 carbon atoms concentration (mg/1) - methane rate production (1 CH₄/1_digester.d) - Km saturation coefficient (mg/l) - Ki inhibition coefficient (mg/l) - specific growth rate (1/d) - maximum specific growth rate (1/d) - b biological decay coefficient (1/d) - Arrhénius coefficient (–) - yield of acidogenic bacteria per mg of biodegradable matter consumed (mg S₁/mg L_b) | (mg S₂/mg VA2) - yield of methanogenic bacteria per mg of VA2 consumed - yield of methane production per mg of S₂ formed (1 CH₄/mg S₂) - proportion of VA2 produced per mg of S₁ biosynthetised - proportion of VA3 produced per mg of S₁ biosynthetised - Y_sp volume of CH₄ produced per g of volatile solids eliminated     

Antispacer peptide nucleic acids for sequence-specific CRISPR-Cas9 modulation

Despite the rapid and broad implementation of CRISPR-Cas9-based technologies, convenient tools to modulate dose, timing, and precision remain limited. Building on methods using synthetic peptide nucleic acids (PNAs) to bind RNA with unusually high affinity, we describe guide RNA (gRNA) spacer-targeted, or ‘antispacer’, PNAs as a tool to modulate Cas9 binding and activity in cells in a sequence-specific manner. We demonstrate that PNAs rapidly and efficiently target complexed gRNA spacer sequences at low doses and without design restriction for sequence-selective Cas9 inhibition. We further show that short PAM-proximal antispacer PNAs achieve potent cleavage inhibition (over 2000-fold reduction) and that PAM-distal PNAs modify gRNA affinity to promote on-target specificity. Finally, we apply antispacer PNAs for temporal regulation of two dCas9-fusion systems. These results present a novel rational approach to nucleoprotein engineering and describe a rapidly implementable antisense platform for CRISPR-Cas9 modulation to improve spatiotemporal versatility and safety across applications.