Conformational changes in Arp2/3 complex induced by ATP, WASp-VCA, and actin filaments

We used fluorescence spectroscopy and EM to determine how binding of ATP, nucleation-promoting factors, actin monomers, and actin filaments changes the conformation of Arp2/3 complex during the process that nucleates an actin filament branch. We mutated subunits of Schizosaccharomyces pombe Arp2/3 complex for labeling with fluorescent dyes at either the C termini of Arp2 and Arp3 or ArpC1 and ArpC3. We measured Förster resonance energy transfer (FRET) efficiency (ET_(eff)) between the dyes in the presence of the various ligands. We also computed class averages from electron micrographs of negatively stained specimens. ATP binding made small conformational changes of the nucleotide-binding cleft of the Arp2 subunit. WASp-VCA, WASp-CA, and WASp-actin-VCA changed the ET_(eff) between the dyes on the Arp2 and Arp3 subunits much more than between dyes on ArpC1 and ArpC3. Ensemble FRET detected an additional structural change that brought ArpC1 and ArpC3 closer together when Arp2/3 complex bound actin filaments. VCA binding to Arp2/3 complex causes a conformational change that favors binding to the side of an actin filament, which allows further changes required to nucleate a daughter filament

Fibrinogen regulates the cytotoxicity of mycobacterial trehalose dimycolate, but is not required for cell recruitment, cytokine response, or control of mycobacterial infection

During inflammatory responses and wound healing, the conversion of soluble fibrinogen to fibrin, an insoluble extracellular matrix, long has been assumed to create a scaffold for the migration of leukocytes and fibroblasts. Previous studies concluded that fibrinogen is a necessary cofactor for mycobacterial trehalose 6,6-dimycolate-induced responses, because trehalose dimycolate-coated beads, to which fibrinogen was ad-sorbed, were more inflammatory than those to which other plasma proteins were adsorbed. Herein, we investigate roles for fibrin(ogen) in an in vivo model of mycobacterial granuloma formation and in infection with Mycobacterium tuberculosis, the causative agent of tuberculosis. In wild-type mice, the subcutaneous injection of trehalose dimycolate-coated polystyrene microspheres, suspended within Matrigel, elicited a pyogranulomatous response during the course of 12 days. In fibrinogen-deficient mice, neutrophils were recruited but a more suppurative lesion developed, with the marked degradation and disintegration of the matrix. Compared to that in wild-type mice, the early formation of granulation tissue in fibrinogen-deficient mice was edematous, hypocellular, and disorganized. These deficiencies were complemented by the addition of exogenous fibrinogen. The absence of fibrinogen had no effect on cell recruitment or cytokine production i

Mycobacterial trehalose dimycolate reprograms macrophage global gene expression and activates matrix metalloproteinases.

Trehalose 6,6′-dimycolate (TDM) is a cell wall glycolipid and an important virulence factor of mycobacteria. In order to study the role of TDM in the innate immune response to Mycobacterium tuberculosis, microarray analysis was used to examine gene regulation in murine bone marrow-derived macrophages in response to 90-μm-diameter polystyrene microspheres coated with TDM. A large number of genes, particularly those involved in the immune response and macrophage function, were up- or downregulated in response to these TDM-coated beads compared to control beads. Genes involved in the immune response were specifically upregulated in a myeloid differentiation primary response gene 88 (MyD88)-dependent manner. The complexity of the transcriptional response also increased greatly between 2 and 24 h. Matrix metalloproteinases (MMPs) were significantly upregulated at both time points, and this was confirmed by quantitative real-time reverse transcription-PCR (RT-PCR). Using an in vivo Matrigel granuloma model, the presence and activity of MMP-9 were examined by immunohistochemistry and in situ zymography (ISZ), respectively. We found that TDM-coated beads induced MMP-9 expression and activity in Matrigel granulomas. Macrophages were primarily responsible for MMP-9 expression, as granulomas from neutrophil-depleted mice showed staining patterns similar to that for wild-type mice. The relevance of these observations to human disease is supported by the similar induction of MMP-9 in human caseous tuberculosis (TB) granulomas. Given that MMPs likely play an important role in both the construction and breakdown of tuberculous granulomas, our results suggest that TDM may drive MMP expression during TB pathogenesis

Mycobacterium and the coat of many lipids

Pathogenic Mycobacterium reside inside vacuoles in their host macrophages. These vacuoles fail to fuse with lysosomes yet interact with early endosomes. Glycoconjugates released by the intracellular bacilli traffic through the host cell and are released through exocytosis. These molecules represent both antigens for immune recognition and modulators of immune function. The molecules play key roles in the induction and maintenance of the granuloma, a tissue response that limits bacterial spread yet ensures persistence of the infection

Spontaneous Reorientation Is Guided by Perceived Surface Distance, Not by Image Matching Or Comparison

Humans and animals recover their sense of position and orientation using properties of the surface layout, but the processes underlying this ability are disputed. Although behavioral and neurophysiological experiments on animals long have suggested that reorientation depends on representations of surface distance, recent experiments on young children join experimental studies and computational models of animal navigation to suggest that reorientation depends either on processing of any continuous perceptual variables or on matching of 2D, depthless images of the landscape. We tested the surface distance hypothesis against these alternatives through studies of children, using environments whose 3D shape and 2D image properties were arranged to enhance or cancel impressions of depth. In the absence of training, children reoriented by subtle differences in perceived surface distance under conditions that challenge current models of 2D-image matching or comparison processes. We provide evidence that children’s spontaneous navigation depends on representations of 3D layout geometry.National Institutes of Health (U.S.) (Grant HD 23103

Conformational changes in Arp2/3 complex induced by ATP, WASp-VCA, and actin filaments

We used fluorescence spectroscopy and EM to determine how binding of ATP, nucleation-promoting factors, actin monomers, and actin filaments changes the conformation of Arp2/3 complex during the process that nucleates an actin filament branch. We mutated subunits of Schizosaccharomyces pombe Arp2/3 complex for labeling with fluorescent dyes at either the C termini of Arp2 and Arp3 or ArpC1 and ArpC3. We measured Förster resonance energy transfer (FRET) efficiency (ET_(eff)) between the dyes in the presence of the various ligands. We also computed class averages from electron micrographs of negatively stained specimens. ATP binding made small conformational changes of the nucleotide-binding cleft of the Arp2 subunit. WASp-VCA, WASp-CA, and WASp-actin-VCA changed the ET_(eff) between the dyes on the Arp2 and Arp3 subunits much more than between dyes on ArpC1 and ArpC3. Ensemble FRET detected an additional structural change that brought ArpC1 and ArpC3 closer together when Arp2/3 complex bound actin filaments. VCA binding to Arp2/3 complex causes a conformational change that favors binding to the side of an actin filament, which allows further changes required to nucleate a daughter filament

Close female friendships and knowledge of recommended abortion methods in Nigeria and the Democratic Republic of the Congo among a representative sample of reproductive-aged women

IntroductionThere is a high incidence of unsafe abortion among women in Nigeria and the DRC. Low knowledge of recommended abortion methods [i.e., surgical and medication abortion (MA) pills] is a barrier for women accessing safe abortions. Women often rely on friends for information about abortion methods. Understanding characteristics of women with knowledge of recommended abortion methods, and MA specifically, and how it is influenced by close female friendships may help identify women most at risk of relying on unsafe abortion.MethodsWe used survey data from Performance Monitoring for Action from 11,106 women of reproductive age in Nigeria (April–May 2018) and 3,697 women in Kinshasa and Kongo Central, DRC, (December 2021–April 2022) to produce representative estimates of knowledge of abortion methods at the national and province levels, respectively. We performed bivariate and multivariate logistic regression to determine which characteristics were independently associated with knowing a recommended abortion method, with knowing of MA pills specifically, and to assess our hypothesis that having at least one female confidante would increase one's odds of knowing about these methods.ResultsA minority (26.9%) of women in Nigeria and the majority in Kinshasa (76.7%) and Kongo Central (58.1%) reported having knowledge of at least one recommended abortion method, while knowledge of MA pills was low in all sites. Having at least one close female confidante was associated with increased odds of knowing a recommend abortion method in Nigeria (aOR = 1.50, 95% CI 1.25–1.79) and in Kongo Central (aOR = 2.66, 95% CI 1.40–5.40), and with increased odds of knowing about MA specifically in Kinshasa (aOR = 1.44, 95% CI 1.08–1.93) and Kongo Central (aOR = 3.61, 95% CI 1.28–10.22), but not Nigeria.DiscussionIn legally restrictive contexts where knowledge of recommended abortion methods (particularly medication abortion) is low, having close female friends is related to increased knowledge of recommended abortion methods

Spontaneous sharp bending of DNA: role of melting bubbles

The role of centrally located and distributed base pair mismatches (‘melting bubbles’) on localized bending and stiffness of short dsDNA fragments is evaluated using time-dependent fluorescence lifetime measurements. Distributed melting bubbles are found to induce larger bending angles and decreased levels of stiffness in DNA than centrally located ones of comparable overall size. Our results indicate that spontaneous local opening-up of the DNA duplex could facilitate sharp bending of short DNA strands even in the absence of DNA binding proteins. We also find that the occurrence of two closely spaced melting bubbles will generally be favored when a large energetic barrier must be overcome in forming the desired bent DNA structure

T4 DNA ligase is more than an effective trap of cyclized dsDNA

T4 DNA ligase is used in standard cyclization assays to trap double-stranded DNA (dsDNA) in low-probability, cyclic or highly bent conformations. The cyclization probability, deduced from the relative yield of cyclized product, can be used in conjunction with statistical mechanical models to extract the bending stiffness of dsDNA. By inserting the base analog 2-aminopurine (2-AP) at designated positions in 89 bp and 94 bp dsDNA fragments, we find that T4 DNA ligase can have a previously unknown effect. Specifically, we observe that addition of T4 ligase to dsDNA in proportions comparable to what is used in the cyclization assay leads to a significant increase in fluorescence from 2-AP. This effect is believed to originate from stabilization of local base-pair opening by formation of transient DNA-ligase complexes. Non-specific binding of T4 ligase to dsDNA is also confirmed using fluorescence correlation spectroscopy (FCS) experiments, which reveal a systematic reduction of dsDNA diffusivity in the presence of ligase. ATP competes with regular DNA for non-covalent binding to the T4 ligase and is found to significantly reduce DNA-ligase complexation. For short dsDNA fragments, however, the population of DNA-ligase complexes at typical ATP concentrations used in DNA cyclization studies is determined to be large enough to dominate the cyclization reaction

Reflections [Complete issue : Fourth Quarter 2003]

Publishing History: Print issues of Reflections magazine were published from 1975 to 1999 and its successor, Reflections on Nursing Leadership (RNL) began publication in 2000. RNL migrated to an online format, http://www.reflectionsonnursingleadership.org, in 2006 and continues today. RNL is a member benefit of the Honor Society of Nursing, Sigma Theta Tau International (STTI). The historical print issues have been made openly available. Publishing Frequency: Quarterly until its transition to online. It’s now updated virtually every day. Format: Print, 1975 - 2005; Online, 2006 - present Feature Articles in this Issue: Hog-housed! Magnet Hospitals: Powerful Force for Excellence March to Magnet Nursing in the NIS/CEE Region: Its Changing Face Nursing Education: Global Perspectives Global Challenge: What If...? Space Nursing: Expanding the Horizons Shortage of Nursing Educators? It Shouldn\u27t Come as a Surprise! Coming of Age in Health Care: Changes, Challenges, Choices The ND: Preparing Nurses for Clinical and Educational Leadership This issue of Reflections is fifty-four pages in length and contains information of interest to STTI members