Strategies for signal amplification in nucleic acid detection

Many aspects of molecular genetics necessitate the detection of nucleic acid sequences. Current approaches involving target amplification (in situ PCR, Primed in situ Labeling, Self-Sustained Sequence Replication, Strand Displacement Amplification), probe amplification (Ligase Chain Reaction, Padlock Probes, Rolling Circle Amplification) and signal amplification (Tyramide Signal Amplification, Branched DNA Amplification) are summarized in the present review, together with their advantages and limitations.     

Ultrastructural features of the apical complex, pellicle, and membranes investing the gamonts of Haemogregarina magna (Apicomplexa: Adeleina)

Mature gamonts of Haemogregarina magna lie within a type of parasitophorous vacuole (Pv) apparently unique to the haemogregarines. The cytoplasm of infected erythrocytes was separated from the parasite by two Pv membranes. An additional membrane, coated on both sides with electron-dense material, closely invested the gamonts. The apical complex of the gamonts includes a conoid, two preconoidal rings, and an elaborate polar ring complex. The latter consisted of the polar ring and approximately 78 posteriorly directed, radially arranged, "tine-like" structures which fuse as they merge anteriorly into the polar ring. Freeze fracture replicas demonstrated that the pellicle of gamonts of H. magna was structurally similar to that of other apicomplexans. The closely apposed inner membranes of the pellicle formed plates which were arranged into strips along the long axis of the gamont. Calculations indicated that 13 such strips are found around the circumference of the gamonts with about six subpellicular microtubules associated with the inner surface of each strip. Gamonts of H. magna share many structural similarities with the kinetes, ookinetes, and sporokinetes of other apicomplexans. We propose that the conoid and polar ring complex are fundamental features of all apicomplexan "kinetes."     

Demographic responses of arctic hares Lepus arcticus placed on two predominantly forested islands in Newfoundland

During January-April 1989. we monitored survival, reproduction, and body condition of 19 radio-collared arctic hares Lepus areticus introduced to two predominantly (80%) forested islands. Merchant (66 1 ha) and Burke (82 6 ha), in Placentia Bay, Newfoundland By late April, bone-marrow fat (42 7%) and kidney fat indices were lower than found in populations on the mountain barrens of western Newfoundland However, most island hares gained weight overwinter, and litter sizes (mean. 4 2 in utero) and testis weights (mean, 8 3 g) were as large or larger than recorded from other introduced and natural populations in Newfoundland The distribution of telemetry locations, tracks and feeding sites indicated that hares frequented the scattered barrens (totalling c 30 ha) on both islands in greater proportion than available These results suggest that, in the absence of snowshoe hares and mammalian predators, forested regions interspersed with small patches of barrens can sustain arctic hares     

In vitro processing of a plant pre-mRNA in a HeLa cell nuclear extract.

In order to determine whether there is a general difference in the splicing mechanism of animal and plant pre-mRNAs, we cloned part of the gene for the small subunit of the ribulose 1,5-bisphosphate carboxylase containing both introns into the SP64 vector. RNA was synthesized with SP6 polymerase and used as substrate for in vitro processing in a HeLa cell nuclear splicing extract. Analyses of the processed RNA demonstrate that both introns of the plant pre-mRNA are efficiently removed in an ordered fashion yielding a faithfully ligated mRNA. Two branch points were identified for intron A and three for intron B. The branched nucleotides are adenosine residues in all cases and are located within a distance from the 3' splice site found to be crucial for lariat formation in animal pre-mRNAs. The implications of these results are discussed in light of our previous observation, that a functional pre-mRNA of the human growth hormone gene was not processed in plant tissue in vivo.     

Roux-en-Y Gastric Bypass Alters Brain Activity in Regions that Underlie Reward and Taste Perception

Background

Roux-en-Y gastric bypass (RYGB) surgery is a very effective bariatric procedure to achieve significant and sustained weight loss, yet little is known about the procedure’s impact on the brain. This study examined the effects of RYGB on the brain’s response to the anticipation of highly palatable versus regular food.

Methods

High fat diet-induced obese rats underwent RYGB or sham operation and were then tested for conditioned place preference (CPP) for the bacon-paired chamber, relative to the chow-paired chamber. After CPP, animals were placed in either chamber without the food stimulus, and brain-glucose metabolism (BGluM) was measured using positron emission tomography (μPET).

Results

Bacon CPP was only observed in RYGB rats that had stable weight loss following surgery. BGluM assessment revealed that RYGB selectively activated regions of the right and midline cerebellum (Lob 8) involved in subjective processes related to reward or expectation. Also, bacon anticipation led to significant activation in the medial parabrachial nuclei (important in gustatory processing) and dorsomedial tegmental area (key to reward, motivation, cognition and addiction) in RYGB rats; and activation in the retrosplenial cortex (default mode network), and the primary visual cortex in control rats.

Conclusions

RYGB alters brain activity in areas involved in reward expectation and sensory (taste) processing when anticipating a palatable fatty food. Thus, RYGB may lead to changes in brain activity in regions that process reward and taste-related behaviors. Specific cerebellar regions with altered metabolism following RYGB may help identify novel therapeutic targets for treatment of obesity.     

Biochemical and structural basis for inhibition of Enterococcus faecalis hydroxymethylglutaryl-CoA synthase, mvaS, by hymeglusin

Hymeglusin (1233A, F244, L-659-699) is established as a specific β-lactone inhibitor of eukaryotic hydroxymethylglutaryl-CoA synthase (HMGCS). Inhibition results from formation of a thioester adduct to the active site cysteine. In contrast, the effects of hymeglusin on bacterial HMG-CoA synthase, mvaS, have been minimally characterized. Hymeglusin blocks growth of Enterococcus faecalis. After removal of the inhibitor from culture media, a growth curve inflection point at 3.1 h is observed (vs 0.7 h for the uninhibited control). Upon hymeglusin inactivation of purified E. faecalis mvaS, the thioester adduct is more stable than that measured for human HMGCS. Hydroxylamine cleaves the thioester adduct; substantial enzyme activity is restored at a rate that is 8-fold faster for human HMGCS than for mvaS. Structural results explain these differences in enzyme-inhibitor thioester adduct stability and solvent accessibility. The E. faecalis mvaS-hymeglusin cocrystal structure (1.95 ?) reveals virtually complete occlusion of the bound inhibitor in a narrow tunnel that is largely sequestered from bulk solvent. In contrast, eukaryotic (Brassica juncea) HMGCS binds hymeglusin in a more solvent-exposed cavity.     

Inhibition of TASK-1 potassium channel by phospholipase C

Thetwo-pore-domain K⁺ channel, TASK-1, was recently shown tobe a target of receptor-mediated regulation in neurons and in adrenalglomerulosa cells. Here, we demonstrate that TASK-1 expressed inXenopus laevis oocytes is inhibited by differentCa²⁺-mobilizing agonists. Lysophosphatidic acid, via itsendogenous receptor, and ANG II and carbachol, via their heterologouslyexpressed ANG II type 1a and M₁ muscarinic receptors,respectively, inhibit TASK-1. This effect can be mimicked by guanosine5'-O-(3-thiotriphosphate), indicating the involvementof GTP-binding protein(s). The phospholipase C inhibitor U-73122reduced the receptor-mediated inhibition of TASK-1. Downstream signalsof phospholipase C action (inositol 1,4,5-trisphosphate, cytoplasmicCa²⁺ concentration, and diacylglycerol) do not mediate theinhibition. Unlike the G_q-coupled receptors, stimulation ofthe G_i-activating M₂ muscarinic receptorcoexpressed with TASK-1 results in an only minimal decrease of theTASK-1 current. However, additional coexpression of phospholipaseC-₂ (which is responsive also to G_i-subunits) renders M₂ receptor activation effective.This indicates the significance of phospholipase C activity in thereceptor-mediated inhibition of TASK-1.

     

A Bayesian Interpretation of the Particle Swarm Optimization and Its Kernel Extension

Particle swarm optimization is a popular method for solving difficult optimization problems. There have been attempts to formulate the method in formal probabilistic or stochastic terms (e.g. bare bones particle swarm) with the aim to achieve more generality and explain the practical behavior of the method. Here we present a Bayesian interpretation of the particle swarm optimization. This interpretation provides a formal framework for incorporation of prior knowledge about the problem that is being solved. Furthermore, it also allows to extend the particle optimization method through the use of kernel functions that represent the intermediary transformation of the data into a different space where the optimization problem is expected to be easier to be resolved–such transformation can be seen as a form of prior knowledge about the nature of the optimization problem. We derive from the general Bayesian formulation the commonly used particle swarm methods as particular cases.     

Rapid degradation of pyrogenic carbon

Pyrogenic carbon (PC‐ charcoal, biochar or black carbon) represents a poorly understood component of the global carbon (C) cycle, but one that has considerable potential to mitigate climate change through provision of long‐term soil C sequestration. Mass balance calculations suggest global PC production and stocks are not in balance, indicating a major gap in our understanding of the processes by which PC is re‐mineralized. We collected PC samples derived from the same wood material and exposed to natural environmental conditions for 1 and 11 years. We subjected these materials to repeated laboratory incubation studies at temperatures of up to 60 °C, as ground surface temperatures above 30 °C and up to 60 °C occur regularly over a significant area of the tropics and sub‐tropics. Mineralization rates were not different for the two samples and followed an exponential Arrhenius function that suggest an average turnover time of 67 years for conditions typical of a tropical savannah environment. Microbial biomass as measured by chloroform fumigation and DNA extractions was the same for the two samples, but abiotic CO₂ production was lower for the fresh PC sample than that for the aged sample. Nuclear magnetic resonance spectroscopy, hydrogen pyrolysis and scanning electron microscopy demonstrate that the measured CO₂ production originates dominantly from polycyclic aromatic compounds rather than any minor labile components. Therefore, rapid, sub‐centennial rates of re‐mineralization of PC on the soil surface in tropical and sub‐tropical environments may represent a major and hitherto unidentified mechanism for balancing the PC production at the global scale.     

The new higher level classification of eukaryotes with emphasis on the taxonomy of protists

This revision of the classification of unicellular eukaryotes updates that of Levine et al. (1980) for the protozoa and expands it to include other protists. Whereas the previous revision was primarily to incorporate the results of ultrastructural studies, this revision incorporates results from both ultrastructural research since 1980 and molecular phylogenetic studies. We propose a scheme that is based on nameless ranked systematics. The vocabulary of the taxonomy is updated, particularly to clarify the naming of groups that have been repositioned. We recognize six clusters of eukaryotes that may represent the basic groupings similar to traditional "kingdoms." The multicellular lineages emerged from within monophyletic protist lineages: animals and fungi from Opisthokonta, plants from Archaeplastida, and brown algae from Stramenopiles.