Neocortical Axon Arbors Trade-off Material and Conduction Delay Conservation

The brain contains a complex network of axons rapidly communicating information between billions of synaptically connected neurons. The morphology of individual axons, therefore, defines the course of information flow within the brain. More than a century ago, Ramón y Cajal proposed that conservation laws to save material (wire) length and limit conduction delay regulate the design of individual axon arbors in cerebral cortex. Yet the spatial and temporal communication costs of single neocortical axons remain undefined. Here, using reconstructions of in vivo labelled excitatory spiny cell and inhibitory basket cell intracortical axons combined with a variety of graph optimization algorithms, we empirically investigated Cajal''s conservation laws in cerebral cortex for whole three-dimensional (3D) axon arbors, to our knowledge the first study of its kind. We found intracortical axons were significantly longer than optimal. The temporal cost of cortical axons was also suboptimal though far superior to wire-minimized arbors. We discovered that cortical axon branching appears to promote a low temporal dispersion of axonal latencies and a tight relationship between cortical distance and axonal latency. In addition, inhibitory basket cell axonal latencies may occur within a much narrower temporal window than excitatory spiny cell axons, which may help boost signal detection. Thus, to optimize neuronal network communication we find that a modest excess of axonal wire is traded-off to enhance arbor temporal economy and precision. Our results offer insight into the principles of brain organization and communication in and development of grey matter, where temporal precision is a crucial prerequisite for coincidence detection, synchronization and rapid network oscillations.     

Temporal variation of amphipod assemblages associated with Sargassum filipendula (Phaeophyta) and its epiphytes in a subtropical shore

The phytal assemblages change in response to variation in biological and environmental conditions. In the present study, we evaluated the temporal variation of amphipod assemblages associated with a Sargassum filipendula bed in a subtropical shore, in relation to variation of the host alga, its epiphytes and local environmental conditions. Samples of S. filipendula with associated amphipods, water temperature, water movement and suspended solids were obtained monthly from June 2000 to May 2001. We recorded 24 species of amphipods associated with S. filipendula. Species richness varied throughout the year, with maximum values in October 2000 and minimum in April 2001. Total amphipod density gradually increased during the sampling period, with the highest value in March 2001. Amphipod diversity and evenness were both positively influenced by epiphyte load and negatively by temperature, with higher values during summer months. Total density and tube-builder density were also positively influenced by temperature, whereas nestler density was influenced by epiphyte load. Individual amphipod species showed significant density fluctuations over the year. The canonical correspondence analysis performed explained 88.2% of the variation, with a strong correlation of water movement, temperature and suspended solids with the first axis, and a strong effect of epiphyte load on both the first and the second axes. The temporal structural variation of the studied algal bed strongly influenced amphipod diversity and assemblage composition, possibly through direct and indirect effects.     

Pulmonary VO2 dynamics during treadmill and arm exercise in peripheral arterial disease.

Slowed pulmonary O(2) uptake (Vo(2)) kinetics in peripheral arterial disease (PAD) have been attributed to impaired limb blood flow and/or peripheral muscle metabolic abnormalities. Although PAD results from atherosclerotic occlusive disease in the arteries to the lower extremities, systemic abnormalities affecting whole body O(2) delivery or vascular function in PAD could also partially explain the exercise impairment. To date, the effects of these systemic abnormalities have not been evaluated. To test the hypothesis that the slowed pulmonary Vo(2) kinetics in PAD reflects local and not systemic abnormalities, Vo(2) kinetics were evaluated after the onset of constant-load exercise of the upper and lower limbs in PAD patients and healthy controls (Con). Ten PAD patients and 10 Con without significant cardiopulmonary dysfunction performed multiple transitions from rest to moderate-intensity arm ergometry and treadmill exercise to assess their Vo(2) kinetic responses. Reactive hyperemic (RH) blood flow was assessed in the arms and legs as a measure of endothelial function. Compared with Con, PAD Vo(2) kinetic phase 2 time constants were prolonged during treadmill exercise (PAD 34.3 +/- 9.2 s vs. Con 19.6 +/- 3.5 s; P < 0.01) but not arm exercise (PAD 38.5 +/- 7.5 s vs. Con 32.5 +/- 9.0 s; P > 0.05). RH blood flow was significantly reduced in the legs (PAD 20.7 +/- 8.3 vs. Con 46.1 +/- 17.1 ml.100 ml(-1).min(-1); P < 0.01) and arms of PAD subjects (PAD 34.0 +/- 8.6 vs. Con 50.8 +/- 12.2 ml.100 ml(-1).min(-1); P < 0.01) compared with Con, but RH limb flow was not correlated with arm or treadmill Vo(2) kinetic responses in either group. In summary, slowed pulmonary Vo(2) kinetics in PAD patients occur only with exercise of the lower limbs affected by the arterial occlusive disease process and are not slowed with exercise of the unaffected upper extremities compared with controls. Furthermore, the slowed pulmonary Vo(2) kinetics of the lower extremity could not be explained by any abnormalities in resting cardiac or pulmonary function and were not related to the magnitude of reduction in limb vascular reactivity.     

A synthetic system links FeFe-hydrogenases to essential E. coli sulfur metabolism

Background

FeFe-hydrogenases are the most active class of H₂-producing enzymes known in nature and may have important applications in clean H₂ energy production. Many potential uses are currently complicated by a crucial weakness: the active sites of all known FeFe-hydrogenases are irreversibly inactivated by O₂.

Results

We have developed a synthetic metabolic pathway in E. coli that links FeFe-hydrogenase activity to the production of the essential amino acid cysteine. Our design includes a complementary host strain whose endogenous redox pool is insulated from the synthetic metabolic pathway. Host viability on a selective medium requires hydrogenase expression, and moderate O₂ levels eliminate growth. This pathway forms the basis for a genetic selection for O₂ tolerance. Genetically selected hydrogenases did not show improved stability in O₂ and in many cases had lost H₂ production activity. The isolated mutations cluster significantly on charged surface residues, suggesting the evolution of binding surfaces that may accelerate hydrogenase electron transfer.

Conclusions

Rational design can optimize a fully heterologous three-component pathway to provide an essential metabolic flux while remaining insulated from the endogenous redox pool. We have developed a number of convenient in vivo assays to aid in the engineering of synthetic H₂ metabolism. Our results also indicate a H₂-independent redox activity in three different FeFe-hydrogenases, with implications for the future directed evolution of H₂-activating catalysts.     

Allosteric conversation in the androgen receptor ligand-binding domain surfaces

Androgen receptor (AR) is a major therapeutic target that plays pivotal roles in prostate cancer (PCa) and androgen insensitivity syndromes. We previously proposed that compounds recruited to ligand-binding domain (LBD) surfaces could regulate AR activity in hormone-refractory PCa and discovered several surface modulators of AR function. Surprisingly, the most effective compounds bound preferentially to a surface of unknown function [binding function 3 (BF-3)] instead of the coactivator-binding site [activation function 2 (AF-2)]. Different BF-3 mutations have been identified in PCa or androgen insensitivity syndrome patients, and they can strongly affect AR activity. Further, comparison of AR x-ray structures with and without bound ligands at BF-3 and AF-2 showed structural coupling between both pockets. Here, we combine experimental evidence and molecular dynamic simulations to investigate whether BF-3 mutations affect AR LBD function and dynamics possibly via allosteric conversation between surface sites. Our data indicate that AF-2 conformation is indeed closely coupled to BF-3 and provide mechanistic proof of their structural interconnection. BF-3 mutations may function as allosteric elicitors, probably shifting the AR LBD conformational ensemble toward conformations that alter AF-2 propensity to reorganize into subpockets that accommodate N-terminal domain and coactivator peptides. The induced conformation may result in either increased or decreased AR activity. Activating BF-3 mutations also favor the formation of another pocket (BF-4) in the vicinity of AF-2 and BF-3, which we also previously identified as a hot spot for a small compound. We discuss the possibility that BF-3 may be a protein-docking site that binds to the N-terminal domain and corepressors. AR surface sites are attractive pharmacological targets to develop allosteric modulators that might be alternative lead compounds for drug design.     

Phase-fluorometry study on dielectric relaxation of acrylodan-labeled human serum albumin.

Dielectric relaxation (DR) of acrylodan-labeled human serum albumin (HSA/AC) was studied by phase-fluorometry. A non-monoexponential behavior of both the total fluorescence--and the DR decays has been found. The protein environment of the fluorescent marker shows DR times ranging from the pico to nanosecond timescale. In fluorescence emission decays measured on the red side of the fluorescence spectrum a time constant (<10 ps) affected by a negative preexponential was found supporting the existence of DR of the excited states.     

The history and rationale of using carbonic anhydrase inhibitors in the treatment of peptic ulcers. In memoriam Ioan Puşcaş (1932–2015)

Carbonic anhydrase (CA, EC 4.2.1.1) inhibitors (CAIs) started to be used in the treatment of peptic ulcers in the 1970s, and for more than two decades, a group led by Ioan Pu?ca? used them for this purpose, assuming that by inhibiting the gastric mucosa CA isoforms, hydrochloric acid secretion is decreased. Although acetazolamide and other sulfonamide CAIs are indeed effective in healing ulcers, the inhibition of CA isoforms in other organs than the stomach led to a number of serious side effects which made this treatment obsolete when the histamine H2 receptor antagonists and the proton pump inhibitors became available. Decades later, in 2002, it has been discovered that Helicobacter pylori, the bacterial pathogen responsible for gastric ulcers and cancers, encodes for two CAs, one belonging to the α-class and the other one to the β-class of these enzymes. These enzymes are crucial for the life cycle of the bacterium and its acclimation within the highly acidic environment of the stomach. Inhibition of the two bacterial CAs with sulfonamides such as acetazolamide, a low-nanomolar H. pylori CAI, is lethal for the pathogen, which explains why these compounds were clinically efficient as anti-ulcer drugs. Thus, the approach promoted by Ioan Pu?ca? for treating this disease was a good one although the rationale behind it was wrong. In this review, we present a historical overview of the sulfonamide CAIs as anti-ulcer agents, in memoriam of the scientist who was in the first line of this research trend.     

One axon-multiple functions: Specificity of lateral inhibitory connections by large basket cells

The functional specificity of the projections of single large basket cells of the cat primary visual cortex was studied using novel analytical approaches. The distribution of the labelled axons and that of the target cells were three-dimensionally reconstructed and compared quantitatively to orientation, direction and ocular dominance maps obtained with the intrinsic signal optical imaging technique. Quantitative analysis was carried out (i) for the entire basket cell, (ii) separately, for local and distal projections of the axon and (iii) by dissecting the same axon into two projection fields at the first bifurcation. It was found that although the functional distributions (orientation, direction and ocular dominance) for the entire cell were multi-modal and broadly tuned, individual main branches of the same cell displayed highly specific topography. In the further analysis, 2-dimensional probability density estimates of the target cell distributions revealed clear clustering which may be important for local subfield antagonism. These findings provide support to the idea that the same basket cell mediates several specific receptive field operations depending on the location of the target somata in the functional maps.     

Coupling of Pressure-Induced Structural Shifts to Spectral Changes in a Yellow Fluorescent Protein

X-ray diffraction analysis of pressure-induced structural changes in the Aequorea yellow fluorescent protein Citrine reveals the structural basis for the continuous fluorescence peak shift from yellow to green that is observed on pressurization. This fluorescence peak shift is caused by a reorientation of the two elements of the Citrine chromophore. This study describes the structural linkages in Citrine that are responsible for the local reorientation of the chromophore. The deformation of the Citrine chromophore is actuated by the differential motion of two clusters of atoms that compose the β-barrel scaffold of the molecule, resulting in a slight bending of the β-barrel. The high-pressure structures also show a perturbation of the hydrogen bonding network that stabilizes the excited state of the Citrine chromophore. The perturbation of this network is implicated in the reduction of fluorescence intensity of Citrine. The blue-shift of the Citrine fluorescence spectrum resulting from the bending of the β-barrel provides structural insight into the transient blue-shifting of isolated yellow fluorescent protein molecules under ambient conditions and suggests mechanisms to alter the time-dependent behavior of Citrine under ambient conditions.     

Changes in breath 13CO2/12CO2 consequent to exercise and hypoxia

Because the natural enrichment of carbohydrate with 13C is greater than that of lipid, we hypothesized that the natural enrichment of exhaled CO2 with 13C (EN) could be used to gauge endogenous substrate utilization in exercising human subjects. To test this, EN and the respiratory exchange ratio (R) which equals the respiratory quotient (RQ) in the steady state, were measured simultaneously in seven subjects. Rest and exercise protocols, performed under conditions of room air (sea level) and hypoxic (inspired O2 fraction = 0.15) breathing, were chosen to cause a variety of patterns of oxidative substrate utilization. Work rates were performed both below and above the subject's lactate threshold (LT). Work above the LT was expected to cause the greatest increase in EN reflecting greater utilization of glucose. There was significant intersubject (P less than 0.05) but not intrasubject variability in resting EN. By 40 min of exercise, EN increased significantly (P less than 0.05) over resting values in all exercise protocols during both room air and hypoxia conditions. In the room air studies, we found no difference in EN during the below-LT work, even though there were significant increases in O2 uptake (VO2). In contrast, above-LT work resulted in significantly greater increases in EN by 20 and 40 min of exercise (P less than 0.05). Contrary to our expectations, we observed no separate effect by hypoxia on the EN during exercise. Both EN and R tended to increase from rest to exercise, but during exercise there was no overall correlation between R and the EN. EN reflects changes in endogenous substrate utilization over relatively long periods of time such as at rest, but delays in the appearance of 13CO2 at the mouth due to dilution in body CO2 pools, and possibly isotopic fractionation, preclude the usefulness of EN as an indicator of endogenous fuel mix during short-term exercise.