Bacterial Filamentation Drives Colony Chirality

This is the final version. Available on open access from the American Society for Microbiology via the DOI in this recordChirality is ubiquitous in nature, with consequences at the cellular and tissue scales. As Escherichia coli colonies expand radially, an orthogonal component of growth creates a pinwheel-like pattern that can be revealed by fluorescent markers. To elucidate the mechanistic basis of this colony chirality, we investigated its link to left-handed, single-cell twisting during E. coli elongation. While chemical and genetic manipulation of cell width altered single-cell twisting handedness, colonies ceased to be chiral rather than switching handedness, and anaerobic growth altered colony chirality without affecting single-cell twisting. Chiral angle increased with increasing temperature even when growth rate decreased. Unifying these findings, we discovered that colony chirality was associated with the propensity for cell filamentation. Inhibition of cell division accentuated chirality under aerobic growth and generated chirality under anaerobic growth. Thus, regulation of cell division is intrinsically coupled to colony chirality, providing a mechanism for tuning macroscale spatial patterning. IMPORTANCE Chiral objects, such as amino acids, are distinguishable from their mirror image. For living systems, the fundamental mechanisms relating cellular handedness to chirality at the multicellular scale remain largely mysterious. Here, we use chemical, genetic, and environmental perturbations of Escherichia coli to investigate whether pinwheel patterns in bacterial colonies are directly linked to single-cell growth behaviors. We discover that chirality can be abolished without affecting single-cell twisting; instead, the degree of chirality was linked to the proportion of highly elongated cells at the colony edge. Inhibiting cell division boosted the degree of chirality during aerobic growth and even introduced chirality to otherwise achiral colonies during anaerobic growth. These findings reveal a fascinating connection between cell division and macroscopic colony patterning.National Institutes of Health (NIH)Allen Discovery Center at Stanford University on Systems Modeling of Infectio

Differential regulation of NF-κB activation and function by topoisomerase II inhibitors

BACKGROUND: While many common chemotherapeutic drugs and other inducers of DNA-damage result in both NF-κB nuclear translocation and DNA-binding, we have previously observed that, depending on the precise stimulus, there is great diversity of the function of NF-κB. In particular, we found that treatment of U-2 OS osteosarcoma cells with the anthracycine daunorubicin or with ultraviolet (UV-C) light resulted in a form of NF-κB that repressed rather than induced NF-κB reporter plasmids and the expression of specific anti-apoptotic genes. Anthracyclines such as daunorubicin can induce DNA-damage though inhibiting topoisomerase II, intercalating with DNA and undergoing redox cycling to produce oxygen free radicals. In this study we have investigated other anthracyclines, doxorubicin and aclarubicin, as well as the anthracenedione mitoxantrone together with the topoisomerase II inhibitor ICRF-193, which all possess differing characteristics, to determine which of these features is specifically required to induce both NF-κB DNA-binding and transcriptional repression in U-2 OS cells. RESULTS: The use of mitoxantrone, which does not undergo redox cycling, and the reducing agent epigallocatechingallate (EGCG) demonstrated that oxygen free radical production is not required for induction of NF-κB DNA-binding and transcriptional repression by these agents and UV-C. In addition, the use of aclarubicin, which does not directly inhibit topoisomerase II and ICRF-193, which inhibits topoisomerase II but does not intercalate into DNA, demonstrated that topoisomerase II inhibition is not sufficient to induce the repressor form of NF-κB. CONCLUSION: Induction of NF-κB DNA-binding and transcriptional repression by topoisomerase II inhibitors was found to correlate with an ability to intercalate into DNA. Although data from our and other laboratories indicates that topoisomerase II inhibition and oxygen free radicals do regulate NF-κB, they are not required for the particular ability of NF-κB to repress rather than activate transcription. Together with our previous data, these results demonstrate that the nature of the NF-κB response is context dependent. In a clinical setting such effects could profoundly influence the response to chemotherapy and suggest that new methods of analyzing NF-κB function could have both diagnostic and prognostic value

Inhibition of nuclear factor kappa-B signaling reduces growth in medulloblastoma in vivo

Abstract Background Medulloblastoma is a highly malignant pediatric brain tumor that requires surgery, whole brain and spine irradiation, and intense chemotherapy for treatment. A more sophisticated understanding of the pathophysiology of medulloblastoma is needed to successfully reduce the intensity of treatment and improve outcomes. Nuclear factor kappa-B (NFκB) is a signaling pathway that controls transcriptional activation of genes important for tight regulation of many cellular processes and is aberrantly expressed in many types of cancer. Methods To test the importance of NFκB to medulloblastoma cell growth, the effects of multiple drugs that inhibit NFκB, pyrrolidine dithiocarbamate, diethyldithiocarbamate, sulfasalazine, curcumin and bortezomib, were studied in medulloblastoma cell lines compared to a malignant glioma cell line and normal neurons. Expression of endogenous NFκB was investigated in cultured cells, xenograft flank tumors, and primary human tumor samples. A dominant negative construct for the endogenous inhibitor of NFκB, IκB, was prepared from medulloblastoma cell lines and flank tumors were established to allow specific pathway inhibition. Results We report high constitutive activity of the canonical NFκB pathway, as seen by Western analysis of the NFκB subunit p65, in medulloblastoma tumors compared to normal brain. The p65 subunit of NFκB is extremely highly expressed in xenograft tumors from human medulloblastoma cell lines; though, conversely, the same cells in culture have minimal expression without specific stimulation. We demonstrate that pharmacological inhibition of NFκB in cell lines halts proliferation and leads to apoptosis. We show by immunohistochemical stain that phosphorylated p65 is found in the majority of primary tumor cells examined. Finally, expression of a dominant negative form of the endogenous inhibitor of NFκB, dnIκB, resulted in poor xenograft tumor growth, with average tumor volumes 40% smaller than controls. Conclusions These data collectively demonstrate that NFκB signaling is important for medulloblastoma tumor growth, and that inhibition can reduce tumor size and viability in vivo. We discuss the implications of NFκB signaling on the approach to managing patients with medulloblastoma in order to improve clinical outcomes.</p

Iron Behaving Badly: Inappropriate Iron Chelation as a Major Contributor to the Aetiology of Vascular and Other Progressive Inflammatory and Degenerative Diseases

The production of peroxide and superoxide is an inevitable consequence of aerobic metabolism, and while these particular "reactive oxygen species" (ROSs) can exhibit a number of biological effects, they are not of themselves excessively reactive and thus they are not especially damaging at physiological concentrations. However, their reactions with poorly liganded iron species can lead to the catalytic production of the very reactive and dangerous hydroxyl radical, which is exceptionally damaging, and a major cause of chronic inflammation. We review the considerable and wide-ranging evidence for the involvement of this combination of (su)peroxide and poorly liganded iron in a large number of physiological and indeed pathological processes and inflammatory disorders, especially those involving the progressive degradation of cellular and organismal performance. These diseases share a great many similarities and thus might be considered to have a common cause (i.e. iron-catalysed free radical and especially hydroxyl radical generation). The studies reviewed include those focused on a series of cardiovascular, metabolic and neurological diseases, where iron can be found at the sites of plaques and lesions, as well as studies showing the significance of iron to aging and longevity. The effective chelation of iron by natural or synthetic ligands is thus of major physiological (and potentially therapeutic) importance. As systems properties, we need to recognise that physiological observables have multiple molecular causes, and studying them in isolation leads to inconsistent patterns of apparent causality when it is the simultaneous combination of multiple factors that is responsible. This explains, for instance, the decidedly mixed effects of antioxidants that have been observed, etc...Comment: 159 pages, including 9 Figs and 2184 reference

Cerebral Ketones Detected by 3T MR Spectroscopy in Patients with High-Grade Glioma on an Atkins-Based Diet

Background and purposeKetogenic diets are being explored as a possible treatment for several neurological diseases, but the physiologic impact on the brain is unknown. The objective of this study was to evaluate the feasibility of 3T MR spectroscopy to monitor brain ketone levels in patients with high-grade gliomas who were on a ketogenic diet (a modified Atkins diet) for 8 weeks.Materials and methodsPaired pre- and post-ketogenic diet MR spectroscopy data from both the lesion and contralateral hemisphere were analyzed using LCModel software in 10 patients.ResultsAt baseline, the ketone bodies acetone and β-hydroxybutyrate were nearly undetectable, but by week 8, they increased in the lesion for both acetone (0.06 ± 0.03 ≥ 0.27 ± 0.06 IU, P = .005) and β-hydroxybutyrate (0.07 ± 0.07 ≥ 0.79 ± 0.32 IU, P = .046). In the contralateral brain, acetone was also significantly increased (0.041 ± 0.01 ≥ 0.16 ± 0.04 IU, P = .004), but not β-hydroxybutyrate. Acetone was detected in 9/10 patients at week 8, and β-hydroxybutyrate, in 5/10. Acetone concentrations in the contralateral brain correlated strongly with higher urine ketones (r = 0.87, P = .001) and lower fasting glucose (r = -0.67, P = .03). Acetoacetate was largely undetectable. Small-but-statistically significant decreases in NAA were also observed in the contralateral hemisphere at 8 weeks.ConclusionsThis study suggests that 3T MR spectroscopy is feasible for detecting small cerebral metabolic changes associated with a ketogenic diet, provided that appropriate methodology is used