Determining specific growth rates in different regions of Salmonella typhimurium colonies

Computer-aided image analysis (IA) techniques were used to determine specific growth rates (μ) in different regions of Salmonella typhimurium colonies grown on solid medium at 30°C. Captured transmission images were converted to optical density values, allowing calculation of μ. Data show that growth was exponential throughout the colony during the period studied (between 13 and 21 h after inoculation), but that μ varied from 0·69 to 0·37 h⁻¹ at the periphery and centre, respectively. Results demonstrate regional variation in μ values within colonies, and support conceptual models of colony development. The technique described illustrates that kinetic growth data can be derived within bacterial colonies, and this may lead towards an improved understanding of the growth dynamics of these structures.     

Differential Sensitivities of Fast- and Slow-Cycling Cancer Cells to Inosine Monophosphate Dehydrogenase 2 Inhibition by Mycophenolic Acid

As uncontrolled cell proliferation requires nucleotide biosynthesis, inhibiting enzymes that mediate nucleotide biosynthesis constitutes a rational approach to the management of oncological diseases. In practice, however, results of this strategy are mixed and thus elucidation of the mechanisms by which cancer cells evade the effect of nucleotide biosynthesis restriction is urgently needed. Here we explored the notion that intrinsic differences in cancer cell cycle velocity are important in the resistance toward inhibition of inosine monophosphate dehydrogenase (IMPDH) by mycophenolic acid (MPA). In short-term experiments, MPA treatment of fast-growing cancer cells effectively elicited G0/G1 arrest and provoked apoptosis, thus inhibiting cell proliferation and colony formation. Forced expression of a mutated IMPDH2, lacking a binding site for MPA but retaining enzymatic activity, resulted in complete resistance of cancer cells to MPA. In nude mice subcutaneously engrafted with HeLa cells, MPA moderately delayed tumor formation by inhibiting cell proliferation and inducing apoptosis. Importantly, we developed a lentiviral vector–based Tet-on label-retaining system that enables to identify, isolate and functionally characterize slow-cycling or so-called label-retaining cells (LRCs) in vitro and in vivo. We surprisingly found the presence of LRCs in fast-growing tumors. LRCs were superior in colony formation, tumor initiation and resistance to MPA as compared with fast-cycling cells. Thus, the slow-cycling compartment of cancer seems predominantly responsible for resistance to MPA.     

Gene doctoring: a method for recombineering in laboratory and pathogenic Escherichia coli strains

Background  

Homologous recombination mediated by the λ-Red genes is a common method for making chromosomal modifications in Escherichia coli. Several protocols have been developed that differ in the mechanisms by which DNA, carrying regions homologous to the chromosome, are delivered into the cell. A common technique is to electroporate linear DNA fragments into cells. Alternatively, DNA fragments are generated in vivo by digestion of a donor plasmid with a nuclease that does not cleave the host genome. In both cases the λ-Red gene products recombine homologous regions carried on the linear DNA fragments with the chromosome. We have successfully used both techniques to generate chromosomal mutations in E. coli K-12 strains. However, we have had limited success with these λ-Red based recombination techniques in pathogenic E. coli strains, which has led us to develop an enhanced protocol for recombineering in such strains.     

High-throughput screen identifies disulfiram as a potential therapeutic for triple-negative breast cancer cells: Interaction with IQ motif-containing factors

Triple-negative breast cancer (TNBC) represents an aggressive subtype, for which radiation and chemotherapy are the only options. Here we describe the identification of disulfiram, an FDA-approved drug used to treat alcoholism, as well as the related compound thiram, as the most potent growth inhibitors following high-throughput screens of 3185 compounds against multiple TNBC cell lines. The average IC₅₀ for disulfiram was ~300 nM. Drug affinity responsive target stability (DARTS) analysis identified IQ motif-containing factors IQGAP1 and MYH9 as direct binding targets of disulfiram. Indeed, knockdown of these factors reduced, though did not completely abolish, cell growth. Combination treatment with 4 different drugs commonly used to treat TNBC revealed that disulfiram synergizes most effectively with doxorubicin to inhibit cell growth of TNBC cells. Disulfiram and doxorubicin cooperated to induce cell death as well as cellular senescence, and targeted the ESA⁺/CD24^-/low/CD44⁺ cancer stem cell population. Our results suggest that disulfiram may be repurposed to treat TNBC in combination with doxorubicin.     

Repeated oral administration of capsaicin increases anxiety-like behaviours with prolonged stress-response in rats

This study was conducted to examine the psycho-emotional effects of repeated oral exposure to capsaicin, the principal active component of chili peppers. Each rat received 1 mL of 0.02% capsaicin into its oral cavity daily, and was subjected to behavioural tests following 10 daily administrations of capsaicin. Stereotypy counts and rostral grooming were significantly increased, and caudal grooming decreased, in capsaicin-treated rats during the ambulatory activity test. In elevated plus maze test, not only the time spent in open arms but also the percent arm entry into open arms was reduced in capsaicin-treated rats compared with control rats. In forced swim test, although swimming duration was decreased, struggling increased in the capsaicin group, immobility duration did not differ between the groups. Repeated oral capsaicin did not affect the basal levels of plasma corticosterone; however, the stress-induced elevation of plasma corticosterone was prolonged in capsaicin treated rats. Oral capsaicin exposure significantly increased c-Fos expression not only in the nucleus tractus of solitarius but also in the paraventricular nucleus. Results suggest that repeated oral exposure to capsaicin increases anxiety-like behaviours in rats, and dysfunction of the hypothalamic-pituitary-adrenal axis may play a role in its pathophysiology.     

Intercellular communication in the rat anterior pituitary gland: an in vivo and in vitro study

The concept of "stimulus-secretion coupling" suggested by Douglas and co-workers to explain the events related to monamine discharge by the adrenal medulla (5, 7) may be applied to other endocrine tissues, such as adrenal cortex (36), pancreatic islets (4), and magnocellular hypothalamic neurons (6), which exhibit a similar ion-dependent process of hormone elaboration. In addition, they share another feature, that of joining neighbor cells via membrane junctions (12, 26, and Fletcher, unpublished observation). Given this, and the reports that hormone secretion by the pars distalis also involves a secretagogue-induced decrease in membrane bioelectric potential accompanied by a rise in cellular [Ca++] (27, 34, 41), it was appropriate to test the possibility that cells of the anterior pituitary gland are united by junctions.     

Allosteric control model of bone remodelling containing periodical modes

To help to understand the modelling process that occurs when a scaffold is implanted it is vital to understand the rather complex bone remodelling process prevalent in native bone. We have formulated a mathematical model that predicts osteoactivity both in scaffolds, as well as in bone in vivo and could set a basis for the more detailed allosteric models. The model is extended towards a bio-cybernetic vision of basic multicellular unit (BMU) action, when some of the regulation loops have been modified to reflect the allosteric control mechanisms, developed by Michaels-Menten, Hill, Koshland-Nemethy-Filmer, Monod-Wyman-Changeux. By implementation of this approach a four-dimensional system was obtained that shows steady cyclic behaviour using a wide range of constants with clear biological meaning. We have observed that a local steady state appears as a limiting cycle in multi-dimensional phase space and this is discussed in this paper. Physiological interpretation of this limiting four-dimension cycle possibly related to a conservative-like value has been proposed. Analysis and simulation of the model has shown an analogy between this conservative value, as a kind of substrate-energy regenerative potential of the bone remodelling system with a molecular nature, and to the classical physical value--energy. This dynamic recovery potential is directed against both mechanical and biomechanical damage to the bone. Furthermore, the current model has credibility when compared to the normal bone remodelling process. In the framework of widely recognised Hill mechanisms of allosteric regulation the cyclic attractor, described formerly for a pure cellular model, prevails for different forms of feedback control. This result indicates the viability of the proposed existence of a conservative value (analogous to energy) that characterises the recovery potential of the bone remodelling cycle. Linear stability analysis has been performed in order to determine the robustness of the basic state, however, additional work is required to study a wider range of constants.     

Dissimilatory Fe(III) reduction by an electrochemically active lactic acid bacterium phylogenetically related to Enterococcus gallinarum isolated from submerged soil

AIMS: The isolation and identification of a glucose-oxidizing Fe(III)-reducing bacteria (FRB) with electrochemical activity from an anoxic environment, and characterization of the role of Fe(III) in its metabolism. METHODS AND RESULTS: A Gram-positive (Firmicutes), nonmotile, coccoid and facultative anaerobic FRB was isolated based on its ability to reduce Fe(III). Using the Vitek Gram-positive identification card kit and 16S rRNA gene sequence analysis, the isolate was identified as Enterococcus gallinarum, designated strain MG25. On glucose this isolate produced lactate plus small amounts of acetate, formate and CO2 and its growth rates were similar in the presence and absence of Fe(O)OH. These results suggest that MG25 can couple glucose oxidation to Fe(III) reduction, but without conservation of energy to support growth. Cyclic voltammetry showed that strain MG25 was electrochemically active. CONCLUSIONS: An electrochemically active and FRB, E. gallinarum MG25, was isolated from submerged soil. Fe(III) is used in the bacterial metabolism as an electron sink. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report concerning the electrochemical activity of glucose-oxidizing FRB, E. gallinarum. This organism and others like it could be used as new biocatalysts to improve the performance of a mediator-less microbial fuel cell.     

Novel IL10 gene family associations with systemic juvenile idiopathic arthritis

Juvenile idiopathic arthritis (JIA) is the most common cause of chronic childhood disability and encompasses a number of disease subgroups. In this study we have focused on systemic JIA (sJIA), which accounts for approximately 11% of UK JIA cases. This study reports the investigation of three members of the IL10 gene family as candidate susceptibility loci in children with sJIA. DNA from 473 unaffected controls and 172 patients with sJIA was genotyped for a single nucleotide polymorphism (SNP) in IL19 and IL20 and two SNPs in IL10. We examined evidence for association of the four SNPs by single marker and haplotype analysis. Significant differences in allele frequency were observed between cases and controls, for both IL10-1082 (p = 0.031) and IL20-468 (p = 0.028). Furthermore, examination of the haplotypes of IL10-1082 and IL20-468 revealed greater evidence for association (global p = 0.0006). This study demonstrates a significant increased prevalence of the low expressing IL10-1082 genotype in patients with sJIA. In addition, we show a separate association with an IL20 polymorphism, and the IL10-1082A/IL20-468T haplotype. The two marker 'A-T' haplotype confers an odds ratio of 2.24 for sJIA. This positive association suggests an important role for these cytokines in sJIA pathogenesis.