Legionella pneumophila inhibits acidification of its phagosome in human monocytes.

ABSTRACT We used quantitative fluorescence microscopy to measure the pH of phagosomes in human monocytes that contain virulent Legionella pneumophila, a bacterial pathogen that multiplies intracellularly in these phagocytes. The mean pH of phagosomes that contain live L. pneumophila was 6.1 in 14 experiments. In the same experiments, the mean pH of phagosomes containing dead L. pneumophila averaged 0.8 pH units lower than the mean pH of phagosomes containing live L. pneumophila, a difference that was highly significant (P < 0.01 in all 14 experiments). In contrast, the mean pH of phagosomes initially containing live E. coil, which were then killed by monocytes, was the same as for phagosomes initially containing dead E. coli. The mean pH of L. pneumophila phagosomes in activated monocytes, which inhibit L. pneumophila intracellular multiplication, was the same as in nonactivated monocytes. To simultaneously measure the pH of different phagosomes within the same monocyte, we digitized and analyzed fluorescence images of monocytes that contained both live L. pneumophila and sheep erythrocytes. Within the same monocyte, live L. pneumophila phagosomes had a pH of ~6.1 and sheep erythrocyte phagosomes had a pH of ~5.0 or below

Effects of disorder in location and size of fence barriers on molecular motion in cell membranes

The effect of disorder in the energetic heights and in the physical locations of fence barriers encountered by transmembrane molecules such as proteins and lipids in their motion in cell membranes is studied theoretically. The investigation takes as its starting point a recent analysis of a periodic system with constant distances between barriers and constant values of barrier heights, and employs effective medium theory to treat the disorder. The calculations make possible, in principle, the extraction of confinement parameters such as mean compartment sizes and mean intercompartmental transition rates from experimentally reported published observations. The analysis should be helpful both as an unusual application of effective medium theory and as an investigation of observed molecular movements in cell membranes.Comment: 9 pages, 5 figure

Molecular motion in cell membranes: analytic study of fence-hindered random walks

A theoretical calculation is presented to describe the confined motion of transmembrane molecules in cell membranes. The study is analytic, based on Master equations for the probability of the molecules moving as random walkers, and leads to explicit usable solutions including expressions for the molecular mean square displacement and effective diffusion constants. One outcome is a detailed understanding of the dependence of the time variation of the mean square displacement on the initial placement of the molecule within the confined region. How to use the calculations is illustrated by extracting (confinement) compartment sizes from experimentally reported published observations from single particle tracking experiments on the diffusion of gold-tagged G-protein coupled mu-opioid receptors in the normal rat kidney cell membrane, and by further comparing the analytical results to observations on the diffusion of phospholipids, also in normal rat kidney cells.Comment: 10 pages, 5 figure

A practical laboratory method to determine ceftazidime-avibactam-aztreonam synergy in patients with New Delhi Metallo-beta-lactamase (NDM) producing Enterobacterales infection

Background: In response to infection with New Delhi Metallo-beta-lactamase (NDM) producing Enterobacterales, combination antimicrobial therapy with ceftazidime/avibactam (CAZ/AVI) plus aztreonam (ATM) has been explored. This study evaluated a practical laboratory method of testing for clinically significant synergy between CAZ/AVI+ATM in NDM producing Enterobacterales. Methods: Minimum inhibitory concentration (MIC) of clinical NDM producing isolates were determined for ATM alone and CAZ/AVI+ATM using broth dilution. Restoration of ATM breakpoint following the addition of CAZ/AVI was explored. A CAZ/AVI E-test/ATM disc method was compared to broth dilution. Results: Of 43 isolates, 33/43 (77%) isolates were ATM resistant (median [range] MIC=56 [16 – 512] mg/L). Addition of CAZ/AVI restored the ATM breakpoint (MIC <4mg/L) in 29/33 (89%) of resistant isolates. Overall, the E-test/disc method correlated with findings from broth dilution in 35/43 (81%) of cases. E-test/disc sensitivity was 77% and specificity 85%. Positive predictive value was 92% and negative predictive value 61%. Conclusion: CAZ/AVI+ATM demonstrated significant synergy in most ATM resistant NDM producing Enterobacterales. The E-test/disc method is a quick, reproducible, and reliable method of testing for clinically relevant synergy in the microbiology laboratory

Fibrin regulates neutrophil migration in response to interleukin 8, leukotriene B4, tumor necrosis factor, and formyl-methionyl-leucyl-phenylalanine

We have examined the capacity of four different chemoattractants/cytokines to promote directed migration of polymorphonuclear leukocytes (PMN) through three-dimensional gels composed of extracellular matrix proteins. About 20% of PMN migrated through fibrin gels and plasma clots in response to a gradient of interleukin 8 (IL-8) or leukotriene B4 (LTB4). In contrast, < 0.3% of PMN migrated through fibrin gels in response to a gradient of tumor necrosis factor alpha (TNF) or formyl-methionyl-leucyl-phenylalanine (FMLP). All four chemoattractants stimulated PMN to migrate through gels composed of collagen IV or of basement membrane proteins (Matrigel), or through filters to which fibronectin or fibrinogen had been adsorbed. PMN stimulated with TNF or FMLP adhered and formed zones of close apposition to fibrin, as measured by the exclusion of a 10-kD rhodamine-polyethylene glycol probe from the contact zones between PMN and the underlying fibrin gel. By this measure, IL-8- or LTB4-treated PMN adhered loosely to fibrin, since 10 kD rhodamine-polyethylene glycol permeated into the contact zones between these cells and the underlying fibrin gel. PMN stimulated with FMLP and IL-8, or FMLP and LTB4, exhibited very little migration through fibrin gels, and three times as many of these cells excluded 10 kD rhodamine-polyethylene glycol from their zones of contact with fibrin as PMN stimulated with IL-8 or LTB4 alone. These results show that PMN chemotaxis is regulated by both the nature of the chemoattractant and the composition of the extracellular matrix; they suggest that certain combinations of chemoattractants and matrix proteins may limit leukocyte movements and promote their localization in specific tissues in vivo

Cellular localization, accumulation and trafficking of double-walled carbon nanotubes in human prostate cancer cells

Carbon nanotubes (CNTs) are at present being considered as potential nanovectors with the ability to deliver therapeutic cargoes into living cells. Previous studies established the ability of CNTs to enter cells and their therapeutic utility, but an appreciation of global intracellular trafficking associated with their cellular distribution has yet to be described. Despite the many aspects of the uptake mechanism of CNTs being studied, only a few studies have investigated internalization and fate of CNTs inside cells in detail. In the present study, intracellular localization and trafficking of RNA-wrapped, oxidized double-walled CNTs (oxDWNT–RNA) is presented. Fixed cells, previously exposed to oxDWNT–RNA, were subjected to immunocytochemical analysis using antibodies specific to proteins implicated in endocytosis; moreover cell compartment markers and pharmacological inhibitory conditions were also employed in this study. Our results revealed that an endocytic pathway is involved in the internalization of oxDWNT–RNA. The nanotubes were found in clathrin-coated vesicles, after which they appear to be sorted in early endosomes, followed by vesicular maturation, become located in lysosomes. Furthermore, we observed co-localization of oxDWNT–RNA with the small GTP-binding protein (Rab 11), involved in their recycling back to the plasma membrane via endosomes from the trans-golgi network

On renormalization group flows and the a-theorem in 6d

We study the extension of the approach to the a-theorem of Komargodski and Schwimmer to quantum field theories in d=6 spacetime dimensions. The dilaton effective action is obtained up to 6th order in derivatives. The anomaly flow a_UV - a_IR is the coefficient of the 6-derivative Euler anomaly term in this action. It then appears at order p^6 in the low energy limit of n-point scattering amplitudes of the dilaton for n > 3. The detailed structure with the correct anomaly coefficient is confirmed by direct calculation in two examples: (i) the case of explicitly broken conformal symmetry is illustrated by the free massive scalar field, and (ii) the case of spontaneously broken conformal symmetry is demonstrated by the (2,0) theory on the Coulomb branch. In the latter example, the dilaton is a dynamical field so 4-derivative terms in the action also affect n-point amplitudes at order p^6. The calculation in the (2,0) theory is done by analyzing an M5-brane probe in AdS_7 x S^4. Given the confirmation in two distinct models, we attempt to use dispersion relations to prove that the anomaly flow is positive in general. Unfortunately the 4-point matrix element of the Euler anomaly is proportional to stu and vanishes for forward scattering. Thus the optical theorem cannot be applied to show positivity. Instead the anomaly flow is given by a dispersion sum rule in which the integrand does not have definite sign. It may be possible to base a proof of the a-theorem on the analyticity and unitarity properties of the 6-point function, but our preliminary study reveals some difficulties.Comment: 41 pages, 5 figure