Microbial imprinted polypyrrole/poly(3-methylthiophene) composite films for the detection of Bacillus endospores

The fabrication of Bacillus subtilis endospore imprinted conducting polymer films and subsequent electrochemical detection of bound spores is reported. Imprinted films were prepared by absorbing spores on the surface of glassy carbon electrodes upon which a polypyrrole, followed by a poly(3-methylthiophene), layer were electrochemically deposited. Spore template release was achieved through soaking the modified electrode in DMSO. Binding of endospores to imprinted films could be detected via impedance spectroscopy by monitoring changes in Y' (susceptance) using Mn(II)Cl2 (0.5M pH 3) as the supporting electrolyte. Here, the change in Y' could be correlated to spore densities between 10(4) and 10(7)cfu/ml. More sensitive detection of absorbed spores was achieved by following endospore germination via changes in film charge as measured using cyclic voltammetry. Here, imprinted films were submerged in spore suspensions to permit absorption, heat activated at 70 degrees C for 10 min prior to transferring to an electrochemical cell containing germination activators. By using the assay format it was possible to detect 10(2)cfu/ml. The observed changes in film charge could be attributed to the interaction of the supporting conducting polymer with dipicolinic acid (DPA) and other constituents released from the core in the course of germination. In all cases, it was not possible to regenerate the imprinted films without losing electrode response. In summary, the study has provided proof-of-concept for fabricating microbial imprinted films using conducting polymers.     

Spermidine/spermine N1-acetyltransferase overexpression in kidney epithelial cells disrupts polyamine homeostasis, leads to DNA damage, and causes G2 arrest

Expression of spermidine/spermine N¹-acetyltransferase (SSAT) increases in kidneys subjected to ischemia-reperfusion injury (IRI). Increased expression of SSAT in vitro leads to alterations in cellular polyamine content, depletion of cofactors and precursors of polyamine synthesis, and reduced cell proliferation. In our model system, a >28-fold increase in SSAT levels in HEK-293 cells leads to depletion of polyamines and elevation in the enzymatic activities of ornithine decarboxylase and S-adenosylmethionine decarboxylase, suggestive of a compensatory reaction to increased polyamine catabolism. Increased expression of SSAT also led to DNA damage and G₂ arrest. The increased DNA damage was primarily due to the depletion of polyamines. Other factors such as increased production of H₂O₂ due to polyamine oxidase activity may play a secondary role in the induction of DNA lesions. In response to DNA damage the ATM/ATR Chk1/2 DNA repair and cell cycle checkpoint pathways were activated, mediating the G₂ arrest in SSAT-expressing cells. In addition, the activation of ERK1 and ERK2, which play integral roles in the G₂/M transition, is impaired in cells expressing SSAT. These results indicate that the disruption of polyamine homeostasis due to enhanced SSAT activity leads to DNA damage and reduced cell proliferation via activation of DNA repair and cell cycle checkpoint and disruption of Raf MEK ERK pathways. We propose that in kidneys subjected to IRI, one mechanism through which increased expression of SSAT may cause cellular injury and organ damage is through induction of DNA damage and the disruption of cell cycle. ischemia-reperfusion injury; polyamine depletion; cell proliferation; DNA repair; cell cycle arrest     

Circadian variations in clock gene expression of human bone marrow CD34+ cells

Time-dependent variations in clock gene expression have recently been observed in mouse hematopoietic cells, but the activity of these genes in human bone marrow (BM) has so far not been investigated. Since such data can be of considerable clinical interest for monitoring the dynamics in stem/progenitor cells, the authors have studied mRNA expression of the clock genes hPer1 , hPer2, hCry1, hCry2, hBmal1, hRev-erb alpha, and hClock in human hematopoietic CD34-positive (CD34( +)) cells. CD34(+) cells were isolated from the BM samples obtained from 10 healthy men at 6 times over 24 h. In addition, clock gene mRNA expression was analyzed in the whole BM in 3 subjects. Rhythms in serum cortisol, growth hormone, testosterone, and leukocyte counts documented that subjects exhibited standardized circadian patterns. All 7 clock genes were expressed both in CD34(+) cells and the whole BM, with some differences in magnitude between the 2 cell populations. A clear circadian rhythm was shown for hPer1, hPer2, and hCry2 expression in CD34(+) cells and for hPer1 in the whole BM, with maxima from early morning to midday. Similar to mouse hematopoietic cells, h Bmal1 was not oscillating rhythmically. The study demonstrates that clock gene expression in human BM stem/progenitor cells may be developmentally regulated, with strong or weaker circadian profiles as compared to those reported in other mature tissues.     

Production of an extracellular alkaline metalloprotease from a newly isolated, moderately halophile, Salinivibrio sp. strain AF-2004

An extracellular protease was produced under stress conditions of high temperature and high salinity by a newly isolated moderate halophile, Salinivibrio sp. strain AF-2004 in a basal medium containing peptone, beef extract, glucose and NaCl. A modification of Kunitz method was used for protease assay. The isolate was capable of producing protease in the presence of sodium chloride, sodium sulfate, sodium nitrate, sodium nitrite, potassium chloride, sodium acetate and sodium citrate. The maximum protease was secreted in the presence of 7.5 to 10% (w/v) sodium sulfate or 3% (w/v) sodium acetate (4.6 U ml⁻¹). Various carbon sources including glucose, lactose, casein and peptone were capable of inducing enzyme production. The optimum pH, temperature and aeration for enzyme production were 9.0, 32 °C and 220 rpm, respectively. The enzyme production corresponded with growth and reached a maximum level during the mid-stationary phase. Maximum protease activity was exhibited in the medium containing 1% (w/v) NaCl at 60 °C, with 18% and 41% activity reductions at temperature 50 and 70 °C, respectively. The optimum pH for enzyme activity was 8.5, with 86% and 75% residual activities at pH 10 and 6, respectively. The activity of enzyme was inhibited by EDTA. These results suggest that the protease secreted by Salinivibrio sp. strain AF-2004 is industrially important from the perspectives of its activity at a broad pH ranges (5.0–10.0), its moderate thermoactivity in addition to its high tolerance to a wide range of salt concentration (0–10% NaCl).     

Effect of neohesperidin dihydrochalcone on the activity and stability of alpha‐amylase: a comparative study on bacterial,fungal, and mammalian enzymes

Neohesperidin dihydrochalcone (NHDC) was recently introduced as an activator of mammalian alpha‐amylase. In the current study, the effect of NHDC has been investigated on bacterial and fungal alpha‐amylases. Enzyme assays and kinetic analysis demonstrated the capability of NHDC to significantly activate both tested alpha‐amylases. The ligand activation pattern was found to be more similar between the fungal and mammalian enzyme in comparison with the bacterial one. Further, thermostability experiments indicated a stability increase in the presence of NHDC for the bacterial enzyme. In silico (docking) test locates a putative binding site for NHDC on alpha‐amylase surface in domain B. This domain shows differences in various alpha‐amylase types, and the different behavior of the ligand toward the studied enzymes may be attributed to this fact. Copyright © 2015 John Wiley & Sons, Ltd.     

A quantitative assay for the monitoring of autophagosome accumulation in different phases of the cell cycle

Macroautophagy is a process accompanied by the formation of double-membrane vesicles known as autophagosomes. Although in recently published reviews various methods for the detection of autophagosomes were described, a reliable technique for the automated quantitative evaluation of autophagosome accumulation is still lacking. Here we developed a new assay which is based on the fact that the number of autophagosomes is correlated with the amount of the LC3-II protein, which is specifically associated with autophagosomal membranes. Monitoring of autophagosome: accumulation was performed by extracting the membrane-unbound LC3-I form of the protein from cells, followed by flow cytometric detection of the autophagosomal membrane-associated fraction of LC3-II. This assay could be used for monitoring autophagosomes by flow cytometry utilizing immunostaining with the antibody against the LC3 protein. It is also suitable for analysis of: cells expressing GFP-LC3. We showed that co-staining with propidium iodide allows detection of basal level of autophagosomes in different phases of the cell cycle. Autophagy activators, such as: rapamycin or cell starvation, were able to induce accumulation of autophagosomes in G0/G1, S and G2/M phases. Thus, utilization of this assay simplifies monitoring of autophagosome accumulation induced by different activators or inhibitors of macroautophagy and it is suggested as being useful in the detection of autophagosomes in different phases of the cell cycle.     

Thermal imaging of the periorbital regions during the presentation of an auditory startle stimulus

Infrared thermal imaging of the inner canthi of the periorbital regions of the face can potentially serve as an input signal modality for an alternative access system for individuals with conditions that preclude speech or voluntary movement, such as total locked-in syndrome. However, it is unknown if the temperature of these regions is affected by the human startle response, as changes in the facial temperature of the periorbital regions manifested during the startle response could generate false positives in a thermography-based access system. This study presents an examination of the temperature characteristics of the periorbital regions of 11 able-bodied adult participants before and after a 102 dB auditory startle stimulus. The results indicate that the startle response has no substantial effect on the mean temperature of the periorbital regions. This indicates that thermography-based access solutions would be insensitive to startle reactions in their user, an important advantage over other modalities being considered in the context of access solutions for individuals with a severe motor disability.     

Anti-chemorepulsive effects of vascular endothelial growth factor and placental growth factor-2 in dorsal root ganglion neurons are mediated via neuropilin-1 and cyclooxygenase-derived prostanoid production

Vascular endothelial growth factor (VEGF) displays neurotrophic and neuroprotective activities, but the mechanisms underlying these effects have not been defined. Neuropilin-1 (NP-1) is a receptor for VEGF165 and placental growth factor-2 (PlGF-2), but the role of NP-1 in VEGF-dependent neurotrophic actions is unclear. Dorsal root ganglion (DRG) neurons expressed high levels of NP-1 mRNA and protein, much lower levels of KDR, and no detectable Flt-1. VEGF165 and PlGF-2 promoted DRG growth cone formation with an effect similar to that of nerve growth factor, whereas the Flt-1-specific ligand, PlGF-1, and the KDR/Flt-4 ligand, VEGF-D, had no effect. The chemorepellent NP-1 ligand, semaphorin 3A, antagonized the response to VEGF and PlGF-2. The specific KDR inhibitor, SU5614, did not affect the anti-chemorepellent effects of VEGF and PlGF-2, whereas a novel, specific antagonist of VEGF binding to NP-1, called EG3287, prevented inhibition of growth cone collapse. VEGF stimulated prostacyclin and prostaglandin E2 production in DRG cultures that was blocked by inhibitors of cyclooxygenases; the anti-chemorepellent activities of VEGF and PlGF-2 were abrogated by cyclooxygenase inhibitors, and a variety of prostacyclin analogues and prostaglandins strikingly inhibited growth cone collapse. These findings support a specific role for NP-1 in mediating neurotrophic actions of VEGF family members and also identify a novel role for prostanoids in the inhibition of neuronal chemorepulsion.