The effect of silica fume on biodegradation of cement paste and its capacity to immobilize strontium during exposure to microbial sulfur oxidation

The disposal of low-level radioactive waste containing isotopes such as strontium by immobilization in cement paste has become common practice. However, the stability of cement paste in the environment may be impaired by sulfuric acid produced by sulfur-oxidizing bacteria. Since biodegradation rates in the environment of most radioactive waste burial sites are too low to be measured, determination of the degradation kinetics of cement paste is a difficult task. This study reports on the development of an accelerated biodegradation system for cement pastes in which the cement paste is exposed to a continuous culture of the sulfur-oxidizing bacterium Halothiobacillus neapolitanus. This system facilitated detection of the biodegradation processes in cement paste after as short a time as 15 days. A comparison of the durability of a cement paste blended with silica fume with that of unblended cement paste showed that the silica fume induced an increase in the leaching of Ca⁺² and Si and enhanced weight loss, indicating rapid deterioration in the structural integrity of the cement paste. The leaching of Sr⁺² from the silica fume amended cement paste was slightly reduced as compared with the non amended cement paste, indicating an increase in immobilization of strontium. Nevertheless, our findings do not support the use of silica fume as a suitable additive for immobilization of low-level radioactive waste.     

Establishment of a tobacco BY2 cell line devoid of plant‐specific xylose and fucose as a platform for the production of biotherapeutic proteins

Plant‐produced glycoproteins contain N‐linked glycans with plant‐specific residues of β(1,2)‐xylose and core α(1,3)‐fucose, which do not exist in mammalian‐derived proteins. Although our experience with two enzymes that are used for enzyme replacement therapy does not indicate that the plant sugar residues have deleterious effects, we made a conscious decision to eliminate these moieties from plant‐expressed proteins. We knocked out the β(1,2)‐xylosyltranferase (XylT) and the α(1,3)‐fucosyltransferase (FucT) genes, using CRISPR/Cas9 genome editing, in Nicotiana tabacum L. cv Bright Yellow 2 (BY2) cell suspension. In total, we knocked out 14 loci. The knocked‐out lines were stable, viable and exhibited a typical BY2 growing rate. Glycan analysis of the endogenous proteins of these lines exhibited N‐linked glycans lacking β(1,2)‐xylose and/or α(1,3)‐fucose. The knocked‐out lines were further transformed successfully with recombinant DNaseI. The expression level and the activity of the recombinant protein were similar to that of the protein produced in the wild‐type BY2 cells. The recombinant DNaseI was shown to be totally free from any xylose and/or fucose residues. The glyco‐engineered BY2 lines provide a valuable platform for producing potent biopharmaceutical products. Furthermore, these results demonstrate the power of the CRISPR/Cas9 technology for multiplex gene editing in BY2 cells.     

Ribosome-binding and anti-microbial studies of the mycinamicins, 16-membered macrolide antibiotics from Micromonospora griseorubida

Macrolides have been effective clinical antibiotics for over 70 years. They inhibit protein biosynthesis in bacterial pathogens by narrowing the nascent protein exit tunnel in the ribosome. The macrolide class of natural products consist of a macrolactone ring linked to one or more sugar molecules. Most of the macrolides used currently are semi-synthetic erythromycin derivatives, composed of a 14- or 15-membered macrolactone ring. Rapidly emerging resistance in bacterial pathogens is among the most urgent global health challenges, which render many antibiotics ineffective, including next-generation macrolides. To address this threat and advance a longer-term plan for developing new antibiotics, we demonstrate how 16-membered macrolides overcome erythromycin resistance in clinically isolated Staphylococcus aureus strains. By determining the structures of complexes of the large ribosomal subunit of Deinococcus radiodurans (D50S) with these 16-membered selected macrolides, and performing anti-microbial studies, we identified resistance mechanisms they may overcome. This new information provides important insights toward the rational design of therapeutics that are effective against drug resistant human pathogens.     

Candidate genes for behavioural ecology

In spite of millions of years of evolutionary divergence, the conservation of gene function is common across distant lineages. As such, genes that are known to influence behaviour in one organism are likely to influence similar behaviours in other organisms. Recent studies of the evolution of behaviour and morphological adaptation support this notion. Thus, the candidate gene approach offers great potential to expand our understanding of behavioural ecology. Changes in the expression of candidate genes can reveal their contribution to behavioural variation and/or phenotypic plasticity. Knowledge of gene function also enables experimental manipulation of behaviour in the lab and in the field. The candidate gene approach provides an accessible and useful tool for generating insights about animals that are not typically associated with genetic experimentation.     

Enhancing the take of injected adipose tissue by a simple method for concentrating fat cells

Injection of aspirated fat for the correction of tissue defects is a common procedure in plastic surgery. The reported rates of fat cell survival vary greatly in the medical literature, and different techniques of harvesting, processing, and reinjecting the fat cells are claimed to be responsible for these differences. However, there is no agreement concerning the best way to process the harvested fat before reinjection. The present study was initiated to examine and evaluate the effect of a simple method of isolating the fat particles on the outcome of fat graft survival. In this study, the nude mouse model was used to examine the survival and take of the fat graft concentrated before injection by the cumbersome recommended closed centrifugation technique in comparison with the authors' recommended open method, using an operating room cotton towel as a platform for concentrating the fat cells and separating them from fluids, oil, and debris. One milliliter of concentrated human fat cells preprocessed by towel separation was injected into the nuchal subcutis of 11 nude mice in the study group, and the same amount of fat that was preprocessed by centrifugation was injected into 11 control mice. Injected fat survived in both groups. No significant differences were found regarding fat graft weight and volume, although a tendency for better survival was noticed in the experimental group. Histologic evaluation of the grafts revealed significantly less fibrosis within the study group, meaning that the quality of the fat grafts was better. The authors found this method to be simple, cheap, and friendly to the surgeon in comparison with traditional processing using the centrifuge.     

Bile duct ligation induced acute inflammation up-regulates cyclooxygenase-2 content and PGE2 release in guinea pig gallbladder smooth muscle cell cultures

INTRODUCTION: This study examines hypotheses that BDL induces increased guinea pig gallbladder smooth muscle PGE2 release by up-regulation of COX-2. METHODS: BDL, Sham and Control Hartley guinea pig gallbladders were placed in cell culture, grown to confluence and underwent Western Blot analysis for smooth muscle cell content of COX-1, COX-2, Prostacylin Synthase, actin, caldesmon, vinculin, meta-vinculin and tropomyosin and were assayed for basal release of 6-keto-PGF(1alpha), PGE2 and TxB2 by EIA. RESULTS: BDL did not alter content of smooth muscle cytoskeletal proteins. BDL for 48 h increased smooth muscle cell release of PGE2 and 6-keto-PGF(1alpha) by 3-fold or more when compared to the Control and Sham groups. Western Blot analysis showed increased content of COX-2 in the BDL group. CONCLUSIONS: BDL for 48 h markedly increased endogenous guinea pig smooth muscle cell PG release, which was due to increased COX-2 synthesis.     

Interaction between muscarinic receptor subtype signal transduction pathways mediating bladder contraction

M(3) muscarinic receptors mediate cholinergic-induced contraction in most smooth muscles. However, in the denervated rat bladder, M(2) receptors participate in contraction because M(3)-selective antagonists [para-fluoro-hexahydro-sila-diphenidol (p-F-HHSiD) and 4-DAMP] have low affinities. However, the affinity of the M(2)-selective antagonist methoctramine in the denervated bladder is consistent with M(3) receptor mediating contraction. It is possible that two pathways interact to mediate contraction: one mediated by the M(2) receptor and one by the M(3) receptor. To determine whether an interaction exists, the inhibitory potencies of combinations of methoctramine and p-F-HHSiD for reversing cholinergic contractions were measured. In normal bladders, all combinations gave additive effects. In denervated bladders, synergistic effects were seen with the 10:1 and 1:1 (methoctramine:p-F-HHSiD wt/wt) combinations. After application of the sarcoplasmic reticulum ATPase inhibitor thapsigargin to normal tissue, the 10:1 and 1:1 ratios became synergistic, mimicking denervated tissue. Thus in normal bladders both M(2) and M(3) receptors can induce contraction. In the denervated bladder, the M(2) and the M(3) receptors interact in a facilitatory manner to mediate contraction.     

Inhibition of vesicular uptake of monoamines by hyperforin

Hyperforin is the major active ingredient of Hypericum perforatum (St John's Wort), a traditional antidepressant medication. This study evaluated its inhibitory effects on the synaptic uptake of monoamines in rat forebrain homogenates, comparing the nature of the inhibition at synaptic and vesicular monoamine transporters. A hyperforin-rich extract inhibited with equal potencies the sodium-dependent uptake of the monoamine neurotransmitters serotonin [5-HT], dopamine [DA] and norepinephrine [NE] into rat brain synaptosomes. Hyperforin inhibited the uptake of all three monoamines noncompetitively, in marked contrast with the competitive inhibition exerted by fluoxetine, GBR12909 or desipramine on the uptake of these monoamines. Hyperforin had no inhibitory effect on the binding of [3H]paroxetine, [3H]GBR12935 and [3H]nisoxetine to membrane presynaptic transporters for 5-HT, DA and NE, respectively. The apparent presynaptic inhibition of monoamine uptake could reflect a "reserpine-like mechanism" by which hyperforin induced release of neurotransmitters from synaptic vesicles into the cytoplasm. Thus, we assessed the effects of hyperforin on the vesicular monoamine transporter. Hyperforin inhibited with equal potencies the uptake of the three tritiated monoamines to rat brain synaptic vesicles. Similarly to the synaptosomal uptake, the vesicular uptake was also noncompetitively inhibited by hyperforin. Notably, hyperforin did not affect the direct binding on [3H]dihydrotetrabenazine, a selective vesicular monoamine transporter ligand, to rat forebrain membranes. Our results support the notion that hyperforin interferes with the storage of monoamines in synaptic vesicles, rather than being a selective inhibitor of either synaptic membrane or vesicular monoamine transporters.