Effectiveness of potassium carbonate sesquihydrate to increase dietary cation-anion difference in early lactation cows.

The effect of additional dietary potassium in early lactation dairy cows was evaluated with the addition of potassium carbonate sesquihydrate, which increased dietary K from 1.3 to 2.1% of dry matter (DM) from wk 3 to 12 of lactation. Cows fed potassium carbonate sesquihydrate in the form of DCAD Plus (Church & Dwight Co. Inc., Princeton, NJ) had increased DM intake, milk fat percentage and yield, energy-corrected milk, and efficiency of milk production per unit of DM intake. Milk fat of cows fed higher dietary K had a lower concentration of trans fatty acids, suggesting a role for potassium carbonate sesquihydrate in the rumen in the biohydrogenation processes converting linoleic to stearic acid. Cows fed the diet with 2.1% K had greater apparent balance of K, and no effects were noted on the concentration of blood Mg or amount of fecal Mg. The data support the feeding of greater amounts of K in the early lactation cow.

Prostaglandin E2 and other cyclic AMP elevating agents inhibit interleukin 2 gene transcription by counteracting calcineurin-dependent pathways.

We have previously shown that prostaglandin E2 and other cAMP elevating agents inhibit the nuclear transcription of the human IL-2 gene by interfering with a Ca(2+)-sensitive T cell signal transduction pathway. Calcineurin, a Ca2+/calmodulin-dependent 2B protein phosphatase, is an essential component of the T cell receptor signal transduction pathway leading to IL-2 gene expression. We have therefore tested the hypothesis that this phosphatase may be a target for the inhibitory effects of cAMP on IL-2 gene transcription. We report here that PGE2 markedly reduces the IL-2 promoter activity that is induced by a constitutively active form of calcineurin. In contrast to the complete inhibition of promoter activity produced by the immunosuppressants cyclosporin A and FK-506, this partial block suggests that PGE2 modulates downstream events needed for lymphokine gene activation. Overexpression of calcineurin in Jurkat cells decreases their apparent sensitivity to the inhibitory effects of PGE2 consistent with the fact that this enzyme plays a physiological role in dephosphorylating substrates of cAMP-dependent kinases in several tissues. These results provide evidence that cAMP-dependent pathways may antagonize calcineurin-regulated cascades for T cell activation in vivo, and suggest crosstalk between the Ca2+ and the cAMP signaling pathways during T cell activation.

Calcineurin mediates human tumor necrosis factor alpha gene induction in stimulated T and B cells.

The tumor necrosis factor alpha (TNF-alpha) gene is rapidly transcribed in activated T cells via a calcium-dependent pathway that does not require de novo protein synthesis, but is completely blocked by the immunosuppressive drugs cyclosporin A (CsA) and FK506. Here we show that calcineurin phosphatase activity is both necessary and sufficient for TNF-alpha gene transcription in T cells, and identify the factor that binds to the kappa 3 element of the TNF-alpha gene promoter as the target for calcineurin action. The ability of analogues of CsA and FK506 to block calcineurin phosphatase activity correlates completely with their ability to inhibit induction of TNF-alpha mRNA, induction of a TNF-alpha promoter reporter plasmid in transiently transfected T cells, and induction of the kappa 3 binding factor in an electrophoretic mobility shift assay. Moreover, a cDNA encoding the constitutively active form of calcineurin is sufficient to activate the TNF-alpha promoter and the kappa 3 element. TNF-alpha gene transcription is also highly inducible, CsA-sensitive, and protein synthesis-independent in B cells stimulated through their surface immunoglobulin receptors. Using the panel of CsA and FK506 analogues, we show that calcineurin participates in the induction of TNF-alpha transcription in activated B cells. These results extend our previous demonstration that the kappa 3 binding factor is related to NFATp, the preexisting subunit of nuclear factor of activated T cells, and suggest that calcineurin-mediated modification of the kappa 3 binding factor in T cells is of key importance in the induction of TNF-alpha transcription.

K(alpha) x-ray emission characterization of 100 Hz, 15 mJ femtosecond laser system with high contrast ratio.

We report K(alpha) x-ray production with a high energy (110 mJ per pulse at 800 nm before compression/15 mJ at 400 nm after compression), high repetition rate (100 Hz), and high pulse contrast (better than 10(-9) at 400 nm) laser system. To develop laser-based x-ray sources for biomedical imaging requires to use high-energy and high-power ultra-fast laser system where compression is achieved under vacuum. Using this type of laser system, we demonstrate long-term stability of the x-ray yield, conversion efficiency higher than 1.5 x 10(-5) with a Mo target, and the x-ray spot size close to the optical focal spot. This high-repetition K(alpha) x-ray source can be very useful for x-ray phase-contrast imaging.

Effect of cobalt supplementation during late gestation and early lactation on milk and serum measures.

Thirty-six multiparous cows were assigned to a study to determine the effects of dietary Co supplementation during late gestation and early lactation on concentrations of Co in serum and liver, vitamin B12 concentrations in serum and milk, and milk yield. Nonlactating cows received diets containing 0.15, 0.89, or 1.71 mg/ kg of Co (dry matter basis) from 55 d before parturition, and lactating cows received diets containing 0.19, 0.57, or 0.93 mg/kg of Co (dry matter basis) from parturition through 120 d postpartum. Serum vitamin B12 concentrations declined sharply in all cows between 55 and 20 d prepartum. Dietary Co supplementation tended to cause an increase in the concentration of vitamin B12 in colostrum and milk. Cobalt intake did not affect concentrations of Co in liver or serum, but increased the Co concentration of milk (0.089, 0.120, and 0.130 microg of Co/mL) at 120 days in milk. There was no effect of Co supplementation on dry matter intake or yield of milk and milk components. In conclusion, serum concentrations of vitamin B12 are reduced in the early dry period, and added dietary Co may increase ruminal synthesis of vitamin B12 as indicated by a tendency for increased vitamin B12 concentrations in colostrum and milk of cows supplemented with dietary Co.

Calcineurin is associated with the cytoskeleton of cultured neurons and has a role in the acquisition of polarity.

Calcineurin is a calmodulin-dependent serine-threonine phosphatase found in many cell types but most abundant in neurons. To determine its localization in developing neurons, dissociated cultures from embryonic day 15 rat cerebellum were analyzed immunocytochemically after treatment with cytoskeletal-disrupting drugs. During the initial outgrowth of neurites, calcineurin is enriched in growth cones where its localization depends upon the integrity of both microtubules and actin filaments. Treatment with cytochalasin shifts calcineurin from the growth cone to the neurite shaft, and with nocadozole calcineurin translocates to the cell body. Therefore calcineurin is well positioned to mediate interactions between cytoskeletal systems during neurite elongation. By 14 d in culture, when the neurons have developed extensive neuronal contacts and synapses are present, calcineurin is predominantly in the neurite shaft. Incubation of cultured cells with Cyclosporin A or a specific peptide, both of which selectively inhibit calcineurin's phosphatase activity, prevented axonal elongation. Because the microtubule-associated protein tau appears to play a key role in asymmetric neurite elongation, we examined modifications in its phosphorylation state resulting from calcineurin inhibition. In contrast to the normal development of cerebellar macroneurons in which reactivity with the phosphorylation-dependent antibody, tau-1, progressively increases, there was a persistent inhibition of tau-1 reactivity in cells exposed to Cyclosporin A. These findings suggest a role for calcineurin in regulating tau phosphorylation and possibly modulating other steps required for the determination of polarity.

A novel use of QR code stickers after orthopaedic cast application.

INTRODUCTION We present a novel solution to ensure that information and contact details are always available to patients while in cast. An information sticker containing both telephone numbers and a Quick Response (QR) code is applied to the cast. When scanned with a smartphone, the QR code loads the plaster team's webpage. This contains information and videos about cast care, complications and enhancing recovery. METHODS A sticker was designed and applied to all synthetic casts fitted in our fracture clinic. On cast removal, patients completed a questionnaire about the sticker. A total of 101 patients were surveyed between November 2015 and February 2016. The questionnaire comprised ten binary choice questions. RESULTS The vast majority (97%) of patients had the sticker still on their cast when they returned to clinic for cast removal. Eighty-four per cent of all patients felt reassured by the presence of the QR code sticker. Nine per cent used the contact details on the cast to seek advice. Over half (56%) had a smartphone and a third (33%) of these scanned the QR code. Of those who scanned the code, 95% found the information useful. CONCLUSIONS This study indicates that use of a QR code reassures patients and is an effective tool in the proactive management of potential cast problems. The QR code sticker is now applied to all casts across our trust. In line with NHS England's Five Year Forward View calling for enhanced use of smartphone technology, our trust is continuing to expand its portfolio of patient information accessible via QR codes. Other branches of medicine may benefit from incorporating QR codes as portals to access such information.

Prediction and evaluation of urine and urinary nitrogen and mineral excretion from dairy cattle.

Urine excretion is a substantial factor in the amount of manure that needs to be managed, and urinary N can contribute to ammonia volatilization. Development and validation of prediction equations focusing on dietary factors to decrease urine and urinary nutrient excretion will provide information for managing urine and feces separately or for other future technologies. The objective of this study was to develop equations for prediction of urine excretion and excretion of urinary N, Na, and K and to evaluate both new and previously published prediction equations for estimation of urine and urinary nutrient excretion from lactating dairy cows. Data sets from metabolism studies conducted at Washington State University were compiled and evaluated for excretion of minerals. Urine excretion averaged 24.1 kg/d and urinary nitrogen excretion ranged from 63 to 499 g/d in the calibration data set. Regression equations were developed to predict urine excretion, urinary N excretion, and urinary Na and K excretion. Predictors used in the regression equations included milk yield, body weight, dietary crude protein percentage, milk urea nitrogen, and nutrient intakes. Previously published prediction equations were evaluated using data sets from Washington State University and the University of Wisconsin. Mean and linear biases were evaluated by determining the regression of residuals on predicted values. Evaluation and validation of prediction equations are important to develop equations that will more accurately estimate urine and urinary nitrogen excretion from lactating dairy cows.

Molecular cloning of a full-length cDNA encoding the catalytic subunit of human calmodulin-dependent protein phosphatase (calcineurin A alpha).

A complementary DNA for human calcineurin A alpha (protein phosphatase-2B), encoding a protein of 521 amino acids, was isolated from a hippocampus library. The deduced human sequence differs from that of mouse in only two amino acids, demonstrating that the structure of this catalytic subunit has been strictly conserved during mammalian evolution. Such high homology is in contrast to that seen for calcineurin A gamma, an isoform that shows only 88% identity between human and mouse (Muramatsu, T. and Kincaid, R.L. (1992) Biochem. Biophys. Res. Commun. 188, 265-271).

Cyclic 3',5'-AMP phosphodiesterase in Physarum polycephalum. I. Chemotaxis toward inhibitors and cyclic nucleotides.

The effect of several inhibitors of the enzyme cyclic 3',5'-AMP phosphodiesterase as chemoattractants in Physarum polycephalum was examined. Of the compounds tested, 4-(3-butoxy-4-methoxybenzyl)-2-imidazolidinone (Roche 20-1724/001) and 1-ethyl-4-(isopropylidinehydrazino)-1H-pyrazolo-(3,4-b)-pyridine-5-carboxylic acid ethyl ester, hydrochloride (Squibb 20009) were the most potent attractants. 3-Isobutyl-1-methyl xanthine, theophylline, and morin (a flavanoid) were moderate attractants and sometimes gave negative chemotaxis at high concentrations. Cyclic 3',5'-AMP was an effective, but not potent attractant. A repellent effect following the positive chemotactic action was sometimes observed with cyclic 3',5'-AMP at concentrations as high as 1 . 10(-2) M. Dibutyryl cyclic AMP appeared to be a somewhat more potent attractant than cyclic 3',5'-AMP. The 8-thiomethyl and 8-bromoderivatives of cyclic AMP, which are poorly hydrolyzed by the phosphodiesterase, were not attractants in Physarum. Possible participation of cyclic 3',5'-AMP in the directional movement in P. polycephalum is discussed.

Cyclic 3',5'AMP phosphodiesterase in Physarum polycephalum. II. Kinetic properties.

Some kinetic characteristics of soluble and particulate cyclic 3',5'-AMP phosphodiesterases from Physarum polycephalum are presented. The nature of enzyme inhibition by various agents is reported. The relationship between various enzyme inhibitors and their reported chemotactic properties in Physarum is examined. The results suggest that the chemoattractant effect of several inhibitors may be related to their ability to inhibit the particulate and extracellular phosphodiesterases, but is unrelated to inhibition of the intracellular enzyme.

Effect of grain source and exogenous phytase on phosphorus digestibility in dairy cows.

Two experiments were conducted to determine P digestibility in lactating dairy cows fed corn or barley as grain sources. The first experiment utilized a replicated incomplete 5 x 4 Latin square design with 8 lactating Holstein cows fed diets containing either corn alone or corn in combination with one of 4 barley varieties that differed in chemical composition. Total tract digestibility of P ranged from 11 to 29% for diets containing the barley varieties and was approximately 35% for the corn diet. A second experiment compared P digestibility in cows fed diets containing corn or barley when exogenous phytase was added to the diets. Lactating Holstein cows (n = 16) were arranged in 4 replications of a Latin square with 2 grains (barley or corn), fed separately or with added exogenous phytase (427 phytase units/kg of total mixed ration and 4 periods of 21 d. Phytate P comprised about 50% of the total P (0.46% P) in the total mixed ration. The concentration of serum inorganic P was higher in cows fed diets with exogenous phytase (5.8 vs. 6.5 mg/dL in cows fed barley diets and 5.5 vs 6.0 mg/dL in cows fed corn diets). Using acid detergent lignin as an internal marker, hydrolysis of phytate P was increased by the exogenous phytase, and total P digestibility tended to be increased. In contrast to Experiment 1, in Experiment 2 there was no effect of grain source on P digestibility and total fecal P. Dry matter intake and efficiency of milk production were not affected by exogenous phytase or grain type. Although phytase activity occurs in the rumen, physical properties of the diet and ruminal passage rates may prevent total hydrolysis of phytate in the rumen of lactating cows. Thus, exogenous dietary phytase might improve P digestibility in dairy cows in some dietary situations.

Prediction of manure and nutrient excretion from dairy cattle.

Accurate estimates of manure excretion are needed for planning manure storage facilities and for nutrient management. Data sets from metabolism studies conducted at several universities were compiled and evaluated for excretion of total manure, N, P, and K. Animal groups included calves weighing up to 204 kg, heifers weighing between 274 and 613 kg, nonlactating cows, and lactating cows. Regression equations were developed to predict excretion of total manure, total dry matter, N, P, and K. Predictors used in the regression equations for lactating cows included milk yield, percentages of protein and fat in milk, dietary concentrations of crude protein and neutral detergent fiber, and intakes of nutrients. The regression equations provide improved predictions of excretion and enable more accurate planning of manure storage and nutrients to be managed at the farm level.

Studies into using manure in a biorefinery concept.

Animal manure is an underutilized biomass resource containing a large amount of organic carbon that is often wasted with the existing manure disposal practices. A research project funded by the US Department of Energy explored the feasibility of using manure via the sugar platform in a biorefinery, converting the carbon from fiber to biochemicals. The results showed that (1) fiber was the major component of manure dry material making up approx 50%, 40%, and 36% of the dry dairy, swine, and poultry manure material, respectively; within dairy manure, more than 56% of the dry matter was in particles larger than 1.680 mm; (2) in addition to being a carbon source, manure could provide a variety of nutrient for fungi T. reesei and A. phoenicis to produce cellulase; (3) the hemicellulose component in the manure fiber could be readily converted to sugar through acid hydrolysis; while concentrated acid decrystallization treatment was most effective in manure cellulose hydrolysis; (4) purification and separation was necessary for further chemical conversion of the manure hydrolysate to polyols through hydrogenation; and (5) the manure utilization strategy studied in this work is currently not profitable.

Identification of calmodulin-binding proteins.

We have outlined and partially characterized a series of biotinylated calmodulin derivatives that may be useful in the study of calmodulin-binding protein expression, physical points of calmodulin-target interaction, and proteolytic mapping of related calmodulin-binding proteins. Biotinylated calmodulins offer several advantages as probes of protein-protein interactions. First, biotinylation can be directed to different amino acid residues. Second, biotinylation can be carried out under mild, near-physiological conditions, reducing the likelihood that conditions of protein modification would destroy biological function. Third, biotinylated proteins are stable, and reagents needed for their preparation and detection are relatively inexpensive. Fourth, the sensitivity of avidin-chromogenic enzyme systems is approaching that of radioactivity, with the added advantage that chromogens can be visualized in a relatively short time with respect to autoradiography. However, as with any protein modification procedure, one must be cautious when interpreting the results obtained with biotinylated proteins. For calmodulin-binding proteins, some interactions are impaired by modification of specific lysyl residues. On the other hand, interaction of biotinylated calmodulin with phosphodiesterase occurs, but this interaction may obscure recognition of the biotin residue by avidin. One approach to circumvent this problem is to have a series of site-directed biotinylated proteins available for use as outlined in this chapter. The choice of which agent to use is determined by the primary sequence of the protein of interest and whether any information is available concerning the effects of chemical modification on structure (i.e., acetylation experiments, modification of free sulfhydryls). In the absence of such information, an empirical approach can be taken. Photobiotin affords an easy means for biotinylation of proteins; however, the sites of modification are not always predictable. NHS-biotin derivatives are readily available and are relatively easy to use. Finally, one may wish to biotinylate the protein while liganded to its normal interacting molecule, in the case of calmodulin, calcium ion is the obvious choice. However, calmodulin could also be biotinylated while bound to a specific binding protein such as calcineurin. The latter method may be of use in determination of changes in reactivities of specific amino acid residues subsequent to binding. Finally, it may prove advantageous to biotinylate genetically engineered calmodulin, yeast calmodulin, or plant calmodulin to further define calmodulin-target protein interactions. Thus, the use of biotinylated calmodulin derivatives may offer insights into a range of structural and functional questions relevant to regulation of specific calmodulin-binding proteins.

Electron microscopic immunocytochemical evidence that the calmodulin-dependent cyclic nucleotide phosphodiesterase is localized predominantly at postsynaptic sites in the rat brain.

The calmodulin-dependent cyclic nucleotide phosphodiesterase represents an important junction between the Ca2+ and the cyclic AMP/cyclic GMP second messenger systems. In brain it is a major cyclic nucleotide-degrading activity and is selectively expressed in the soma and dendrites of regional output neurons [Kincaid et al. (1987) Proc. natn. Acad. Sci. U.S.A. 84, 1118-1122]. In this study the subcellular localization of this enzyme in cerebral cortex, hippocampus and inferior colliculus of rat brain was analysed by electron microscopic immunocytochemical methods using affinity-purified antibodies. The immunoreactivity was found exclusively within neurons whereas glial cells were unstained; preabsorption of antibody with phosphodiesterase eliminated this reactivity, demonstrating the specificity of immunostaining. In the neuronal cell bodies, deposits of immunoreaction product occurred as sparse patches in the cytoplasm and were often associated with organelles such as mitochondria, Golgi-complex and endoplasmic reticulum; nuclei, however, were free from immunoreaction product. In the neuronal processes immunoreactivity was found within dendrites and dendritic spines, whereas the myelinated axons and axon terminals were immunonegative. The postsynaptic densities of asymmetric synapses were associated with especially high concentrations of immunoreaction product. However, the immunopositive synaptic profiles appeared to be quite selective, comprising only a small percentage of the total number of synapses in the neuropil. Our results indicate that the calmodulin-dependent cyclic nucleotide phosphodiesterase is concentrated at postsynaptic sites in specific classes of neurons. This finding supports other morphological evidence indicating a primary role for cyclic nucleotide action in postsynaptic and not presynaptic structures. Furthermore, since this enzyme is regulated by Ca2+, this interface between second messenger systems seems to play a significant role in the postsynaptic integration of Ca(2+)-mediated neuronal inputs.

A rapid non-radioactive procedure for plaque hybridization using biotinylated probes prepared by random primed labeling.

A simple, non-radioactive method has been developed for the rapid screening of phage libraries. In the present study, nanogram amounts of a small restriction fragment (135 bp) were biotinylated via random primed labeling and used to probe cDNA libraries using a modified plaque hybridization protocol. The high backgrounds that are seen typically with avidin/biotin-based methods for plaque hybridization were eliminated by incubation of filters with one of several different proteases prior to hybridization. A comparison of several detection systems indicated that streptavidin conjugated to calf intestinal alkaline phosphatase (AP) was the most sensitive, yielding signals comparable to those obtained with 32P-labeled probes. The times required for phage growth and pre-hybridization were reduced substantially, permitting a convenient one-day screening protocol. Nitrocellulose filters gave the best signal to noise ratio, although "streaking" of plaque DNA was observed occasionally; this problem can be overcome by using nylon-based membranes, which allows exact visualization of the positive plaques. The method was highly reliable; 29 out of 33 putative clones retested positive and the authenticity of these was confirmed by DNA sequence analysis. The random primed biotinylation procedure has been utilized successfully with several different cDNA fragments and has proven useful for other hybridization-based methods (Northern and Southern blots), without the problems associated with the use of radiolabeled probes.

Differential subcellular localization of neural isoforms of the catalytic subunit of calmodulin-dependent protein phosphatase (calcineurin) in central nervous system neurons: immunohistochemistry on formalin-fixed paraffin sections employing antigen retrieval by microwave irradiation.

We examined the immunohistochemical distribution of the two mammalian isoforms of calcineurin catalyic subunits, A alpha and A beta, in central nervous system (CNS) tissues of cows, rats, and humans. Cryostat sections and paraffin sections of parformaldehyde-fixed tissues were stained with antipeptide antibodies for each isoform. The same localization pattern was observed in both cryostat and paraffin sections. In the latter, the intensity of the staining was dramatically enhanced by microwave irradiation. Calcineurin isoforms were localized in a variety of nerve cells but not in neuroglial cells. Their differential expression as the A alpha isoform in the nucleus and the A beta isoform in the cytoplasm was present in a variety of CNS nerve cells, most distinctively in Purkinje cells of the cerebellum and pyramidal cells of the cerebrum, irrespective of species. These results suggest that each isform has distinct intracellular sites of action in CNS neurons and that the phenomenon has been conserved during mammalian evolution.

Molecular and phylogenetic analysis of calmodulin-dependent protein phosphatase (calcineurin) catalytic subunit genes.

In the mammalian brain, there are multiple catalytic subunits for the Ca(2+)- and calmodulin-dependent protein phosphatase [also called protein phosphatase 2B (PP-2B) and calcineurin] that are derived from two structural genes. The coding sequences of these two genes are distinguished by the absence (PP2B alpha 1) or the presence (PP2B alpha 2) of an amino terminus containing polyproline. Both of these genes can produce intragenic isoforms through alternative splicing. In the present study, a potential phylogenetic relationship of these genes was inferred from analysis of genomic DNA and from studies of mRNA and protein expression. Southern blot analysis showed unique restriction fragments for both genes in seven mammalian species; however, in organisms from two nonmammalian vertebrates (chicken and lizard), hybridization was observed only for PP2B alpha 1. In agreement with these results, Northern blots of mammalian brain RNA showed transcripts for both genes, with about two to three times more of the PP2B alpha 1 mRNAs, whereas in chicken and lizard, only PP2B alpha 1 transcripts were detected. An analysis of protein expression by two-dimensional electrophoresis was also consistent with these findings. For the purified mammalian brain protein, eight to ten variants were observed with isoelectric points of 5.2-5.8; immunoblot analysis using anti-peptide antibodies indicated that the majority of these were PP2B alpha 1 forms. In chicken brain, multiple isoforms were recognized by antibodies against the PP2B alpha 1 forms, but no reactivity was seen with those against the PP2B alpha 2 forms. Taken together, these findings suggest that: (i) in mammals, the predominant catalytic subunit isoforms in brain are PP2B alpha 1 products and (ii) the gene for the polyproline-containing catalytic subunit of calmodulin-dependent phosphatase (PP2B alpha 2) may have evolved after the avian/reptilian branching point, perhaps to carry out a role(s) of particular significance in mammals.

In vivo calcineurin crystals formed using the baculovirus expression system.

Calcineurin is a heterodimeric phosphatase involved in the signal transduction of antigen-activated T cells. Coexpression of its two subunits, the regulatory subunit from human and the catalytic subunit from Neurospora crassa in cultured insect cells using the baculovirus expression system results in the formation of very large crystals in the cytoplasm. The crystals are formed initially in vesicles, but their subsequent growth appears to be uninhibited and continues without the need of an enclosing membrane until the host cell lyses. Although these in vivo crystals are low in population, ranging only 0-3 per cell, they are extremely large, over 10 mu m in some cases. Biochemical assays confirm their calcineurin origin, with the regulatory subunit incorporated being myristoylated, although both the myristoylated and unmyristoylated forms are expressed. The lattice structure of the in vivo crystals, with a spacing of 5.5 nm, is preserved with the regular electron microscopic (EM) specimen preparation procedure.