Tissue Molds for Cryostat Embedding

Ethidium bromide, (0.1% solution in ethanol-water, 1:3, v/v) was used to stain nuclei in mycelia and spores of different fungi. Nuclei looked bright brick red under green excitation. This method is very efficient, specific, reproducible and cost-effective.     

Aluminum Foil Molds for Cryostat Blocks

Inexpensive pyramidal templates may be prepared in a range of sizes from segments of plastic or wooden bars (Fig. 1). Pieces of double-layered aluminum cooking foil are shaped by hand around templates of appropriate size and their surfaces are smoothed with fingernails. In this way, “negative” molds of known cross-section and variable depth can be obtained (Fig. 1). Tissue samples are oriented on the bottom of the molds in a few drops of Ames O.C.T. Compound and just covered with the same embedding medium. Narrow strips of light-weight cardboard with identification data are inserted into the blocks close to a lateral surface. The blocks are solidified on the cold stage of a cryostat (a drop of O.C.T. will improve thermal contact and help keep them upright), or with a liquid gas, such as nitrogen, or Freon 22 (in the latter case the molds are immersed as far as the upper level of the O.C.T., but are not submerged, so that the Freon does not bubble into the liquid O.C.T.). After freezing, the foil is removed and the blocks, handled with cold forceps, are mounted on cryostat chucks with O.C.T.     

Application of Artificial Neural Networks to Investigate One-Carbon Metabolism in Alzheimer’s Disease and Healthy Matched Individuals

Folate metabolism, also known as one-carbon metabolism, is required for several cellular processes including DNA synthesis, repair and methylation. Impairments of this pathway have been often linked to Alzheimer’s disease (AD). In addition, increasing evidence from large scale case-control studies, genome-wide association studies, and meta-analyses of the literature suggest that polymorphisms of genes involved in one-carbon metabolism influence the levels of folate, homocysteine and vitamin B12, and might be among AD risk factors. We analyzed a dataset of 30 genetic and biochemical variables (folate, homocysteine, vitamin B12, and 27 genotypes generated by nine common biallelic polymorphisms of genes involved in folate metabolism) obtained from 40 late-onset AD patients and 40 matched controls to assess the predictive capacity of Artificial Neural Networks (ANNs) in distinguish consistently these two different conditions and to identify the variables expressing the maximal amount of relevant information to the condition of being affected by dementia of Alzheimer’s type. Moreover, we constructed a semantic connectivity map to offer some insight regarding the complex biological connections among the studied variables and the two conditions (being AD or control). TWIST system, an evolutionary algorithm able to remove redundant and noisy information from complex data sets, selected 16 variables that allowed specialized ANNs to discriminate between AD and control subjects with over 90% accuracy. The semantic connectivity map provided important information on the complex biological connections among one-carbon metabolic variables highlighting those most closely linked to the AD condition.     

Lactate Metabolism and Its Effects on Glucose Metabolism in an Excised Neural Tissue

Abstract: Chains of lumbar sympathetic ganglia, excised from 15-day-old chicken embryos, were incubated for 4 h at 36°C in a bicarbonate-buffered physiological salt solution containing 5.5 mM glucose and equilibrated with 5% CO₂–95% O₂. [U-¹⁴C]Glucose and [U-¹⁴C]lactate were used as tracers to measure the products of glucose and lactate metabolism, respectively, including CO₂, lactate, and constituents of the tissue. When 5 mM lactate was added to bathing solution containing 5.5 mM glucose, lactate carbon displaced 50–70% of the glucose carbon otherwise used for CO₂ production and provided about three times as much carbon for CO₂ as did glucose. The lactate addition increased the total carbon incorporated into CO₂ and into constituents of the tissue above those observed with glucose alone and also increased the lactate released to the bathing solution from [U-¹⁴C]-glucose. The latter increase was evidently due to an interference with reuptake of the lactate released from the ganglion cells, not to an increase in the cellular release itself. When the volume of bathing solution was increased 10-fold relative to that of the tissue, the average output of CO₂ from [U-¹⁴C]glucose during a 4-h incubation was decreased by 50% when 5 mM lactate was present but was not affected significantly in the absence of added lactate. It is concluded that the effect of changing volume in the presence of lactate was due to the effects of lactate on glucose metabolism described above and resulted from a lower average lactate concentration in the smaller volume than in the larger one, due to metabolic depletion of the added lactate. Consumable substrates other than lactate, such as glutamine and certain amino acids, also affected glucose metabolism.     

Silicone Kubber Multiple Molds for Paraffin Embedding

It is frequently desirable, when embedding specimens in paraffin, to take them through the procedure in groups rather than singly. Ackerman (1958) has described a multiple plastic mold which may be used for this purpose, while Adel-son and Schatz (1957) have recommended the use of plastic ice-cube trays. With the first of these there is the advantage that the size and number of compartments can be adjusted to the experimental requirements; with the second, the removal of the hardened blocks from the tray is much more convenient.     

Neural and hormonal control of Brunner's gland secretion

For many years it has been speculated that the physiological function of Brunner's glands was to secrete mucus to protect the proximal duodenum from the corrosive effects of acidified gastric juice. However the control of Brunner's gland secretion remains an enigma. Some evidence exists which indicates both cholinergic and adrenergic innervation of these glands, but current consensus weighs heavily in favor of a hormonal stimulus for glandular secretion. This is based in part on evidence obtained from denervated Brunner's gland pouches following a feeding stimulus. A number of hormones and hormone-like substances have been investigated as possible mediators in this secretory response, however, no specificity was ever demonstrated. The inability to pinpoint a given substance as a common mediator can be attributed to the fact that most active agents employed also affect duodenal motility. We present evidence that Brunner's gland secretion can be observed to be a diphasic response. The initial, transient response is always observed in the presence of increased duodenal motility. The sustained response does not require duodenal motility and is probably hormonally mediated.     

Multicellular Computing Using Conjugation for Wiring

Recent efforts in synthetic biology have focussed on the implementation of logical functions within living cells. One aim is to facilitate both internal “re-programming” and external control of cells, with potential applications in a wide range of domains. However, fundamental limitations on the degree to which single cells may be re-engineered have led to a growth of interest in multicellular systems, in which a “computation” is distributed over a number of different cell types, in a manner analogous to modern computer networks. Within this model, individual cell type perform specific sub-tasks, the results of which are then communicated to other cell types for further processing. The manner in which outputs are communicated is therefore of great significance to the overall success of such a scheme. Previous experiments in distributed cellular computation have used global communication schemes, such as quorum sensing (QS), to implement the “wiring” between cell types. While useful, this method lacks specificity, and limits the amount of information that may be transferred at any one time. We propose an alternative scheme, based on specific cell-cell conjugation. This mechanism allows for the direct transfer of genetic information between bacteria, via circular DNA strands known as plasmids. We design a multi-cellular population that is able to compute, in a distributed fashion, a Boolean XOR function. Through this, we describe a general scheme for distributed logic that works by mixing different strains in a single population; this constitutes an important advantage of our novel approach. Importantly, the amount of genetic information exchanged through conjugation is significantly higher than the amount possible through QS-based communication. We provide full computational modelling and simulation results, using deterministic, stochastic and spatially-explicit methods. These simulations explore the behaviour of one possible conjugation-wired cellular computing system under different conditions, and provide baseline information for future laboratory implementations.     

Cape Town’s Metabolism: Insights from a Material Flow Analysis

This work aims to contribute to the number of urban metabolism case studies using a standardized methodology. An economy‐wide material flow analysis (EW‐MFA) was conducted on the Metropolitan Municipality of Cape Town (South Africa) for the year 2013, using the Eurostat framework. The study provides insights into the city's metabolism through various indicators including direct material input (DMI), domestic material consumption (DMC), and direct material output (DMO), among others. In order to report on the uncertainty of the data, a set of data quality indicators originating from the life cycle assessment literature was used. The results show that domestic extraction involves significant quantities of non‐metallic minerals, and that imports consist primarily of biomass and fossil fuels. The role of the city as a regional hub is also made clear from this study and illustrated by large quantities of food and other materials flowing through the city on their way to or from international markets. The results are compared with indicators from other cities and with previous metabolism work done on Cape Town. To fully grasp the impacts of the city's metabolism, more work needs to be done. It will be necessary to understand the upstream impact of local consumption, and consumption patterns should be differentiated on a more nuanced level (taking into account large differences between household income levels as well as separating the metabolism of industry and commerce from residential consumption).     

Total Wiring Length Minimization of C. elegans Neural Network: A Constrained Optimization Approach

Using the most recent data on the connectivity of the C. elegans neural network, we find optimal two-dimensional positions of interneurons that minimize the total wiring length provided that the positions of motor and sensory neurons are fixed. The rationale behind fixing motor and sensory neurons is the following: while positions of motor and sensory neurons can be influenced by the locations of muscles and sensory organs they are attached to, the main function of interneurons is to connect other neurons, and their placement could try to minimize the wiring length. Solutions for l ¹, l ² and squared l ²–norm were obtained. For the Euclidean norm l ², the relative and absolute difference between the real and optimal total wiring lengths is minimal among these functions of distance. Additional network constraints were discussed such as assignment of different weights to electrical or chemical connections, fixation of “tail” interneurons, minimal interneural distance limitation, and others. These constraints were compared by their influence on the optimal positions of interneurons.     

Noncanonical Modulation of the eIF2 Pathway Controls an Increase in Local Translation during Neural Wiring

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Profile of Nucleosides and Nucleotides in Donkey’s Milk

Nucleotides play a crucial role to cellular functions; they can be obtained from the diet or through the nucleotide salvage pathway, however, in particular situations (occurring mainly in newborns) the metabolic demand of nucleotides exceeds the capacity of their synthesis. These molecules, are receiving attention from a nutraceutical point of view because of their potential direct role in regulating metabolism and infant body condition. Donkey's milk may be considered a good replacer for cow's milk in feeding children with severe Ig-E mediated cow's milk protein allergy, due to its high similarity with human milk. In this study, the presence of cytidine, uridine, CMP, UMP, guanosine, and adenosine, involved in numerous biochemical and physiological activities, were detected for the first time through a RP-HPLC method.     

Cancer & Metabolism

正ORGANIZERS:Navdeep Chandel Northwestern University,USA Hozumi Motohashi Tohoku University,JAPAN Hongyuan Yang University of New South Wales,AUSTRALIA Shimin Zhao Fudan University,CHINA     

Drinking-Straw Molds for Embedding Multiples of Longitudinal Segments for Cryostat Sectioning

It is often desirable to obtain cryostat cross sections of tubular structures, such as oviduct, uterus, esophagus, or small intestine, and to retain the sequential relationships of such sections. In this laboratory the following technic which has been used on rat and mouse tissues from newborn and weanling animals has proved to be rapid, inexpensive, and productive of uniformly excellent results.