50 research outputs found
Structural and functional characterization of endothelial microparticles released by cigarette smoke
Circulating endothelial microparticles (EMPs) are emerging as biomarkers of chronic obstructive pulmonary disease (COPD) in individuals exposed to cigarette smoke (CS), but their mechanism of release and function remain unknown. We assessed biochemical and functional characteristics of EMPs and circulating microparticles (cMPs) released by CS. CS exposure was sufficient to increase microparticle levels in plasma of humans and mice, and in supernatants of primary human lung microvascular endothelial cells. CS-released EMPs contained predominantly exosomes that were significantly enriched in let-7d, miR-191; miR-126; and miR125a, microRNAs that reciprocally decreased intracellular in CS-exposed endothelium. CS-released EMPs and cMPs were ceramide-rich and required the ceramide-synthesis enzyme acid sphingomyelinase (aSMase) for their release, an enzyme which was found to exhibit significantly higher activity in plasma of COPD patients or of CS-exposed mice. The ex vivo or in vivo engulfment of EMPs or cMPs by peripheral blood monocytes-derived macrophages was associated with significant inhibition of efferocytosis. Our results indicate that CS, via aSMase, releases circulating EMPs with distinct microRNA cargo and that EMPs affect the clearance of apoptotic cells by specialized macrophages. These targetable effects may be important in the pathogenesis of diseases linked to endothelial injury and inflammation in smokers
BLUF Domain Function Does Not Require a Metastable Radical Intermediate State
BLUF
(blue light using flavin) domain proteins are an important
family of blue light-sensing proteins which control a wide variety
of functions in cells. The primary light-activated step in the BLUF
domain is not yet established. A number of experimental and theoretical
studies points to a role for photoinduced electron transfer (PET)
between a highly conserved tyrosine and the flavin chromophore to
form a radical intermediate state. Here we investigate the role of
PET in three different BLUF proteins, using ultrafast broadband transient
infrared spectroscopy. We characterize and identify infrared active
marker modes for excited and ground state species and use them to
record photochemical dynamics in the proteins. We also generate mutants
which unambiguously show PET and, through isotope labeling of the
protein and the chromophore, are able to assign modes characteristic
of both flavin and protein radical states. We find that these radical
intermediates are not observed in two of the three BLUF domains studied,
casting doubt on the importance of the formation of a population of
radical intermediates in the BLUF photocycle. Further, unnatural amino
acid mutagenesis is used to replace the conserved tyrosine with fluorotyrosines,
thus modifying the driving force for the proposed electron transfer
reaction; the rate changes observed are also not consistent with a
PET mechanism. Thus, while intermediates of PET reactions can be observed
in BLUF proteins they are not correlated with photoactivity, suggesting
that radical intermediates are not central to their operation. Alternative
nonradical pathways including a keto–enol tautomerization induced
by electronic excitation of the flavin ring are considered
Mechanisms of Size Control and Polymorphism in Viral Capsid Assembly
We simulate the assembly dynamics of icosahedral capsids from subunits that
interconvert between different conformations (or quasi-equivalent states). The
simulations identify mechanisms by which subunits form empty capsids with only
one morphology but adaptively assemble into different icosahedral morphologies
around nanoparticle cargoes with varying sizes, as seen in recent experiments
with brome mosaic virus (BMV) capsid proteins. Adaptive cargo encapsidation
requires moderate cargo-subunit interaction strengths; stronger interactions
frustrate assembly by stabilizing intermediates with incommensurate curvature.
We compare simulation results to experiments with cowpea chlorotic mottle virus
empty capsids and BMV capsids assembled on functionalized nanoparticles and
suggest new cargo encapsidation experiments. Finally, we find that both empty
and templated capsids maintain the precise spatial ordering of subunit
conformations seen in the crystal structure even if interactions that preserve
this arrangement are favored by as little as the thermal energy, consistent
with experimental observations that different subunit conformations are highly
similar
Global patient outcomes after elective surgery: prospective cohort study in 27 low-, middle- and high-income countries.
BACKGROUND: As global initiatives increase patient access to surgical treatments, there remains a need to understand the adverse effects of surgery and define appropriate levels of perioperative care. METHODS: We designed a prospective international 7-day cohort study of outcomes following elective adult inpatient surgery in 27 countries. The primary outcome was in-hospital complications. Secondary outcomes were death following a complication (failure to rescue) and death in hospital. Process measures were admission to critical care immediately after surgery or to treat a complication and duration of hospital stay. A single definition of critical care was used for all countries. RESULTS: A total of 474 hospitals in 19 high-, 7 middle- and 1 low-income country were included in the primary analysis. Data included 44 814 patients with a median hospital stay of 4 (range 2-7) days. A total of 7508 patients (16.8%) developed one or more postoperative complication and 207 died (0.5%). The overall mortality among patients who developed complications was 2.8%. Mortality following complications ranged from 2.4% for pulmonary embolism to 43.9% for cardiac arrest. A total of 4360 (9.7%) patients were admitted to a critical care unit as routine immediately after surgery, of whom 2198 (50.4%) developed a complication, with 105 (2.4%) deaths. A total of 1233 patients (16.4%) were admitted to a critical care unit to treat complications, with 119 (9.7%) deaths. Despite lower baseline risk, outcomes were similar in low- and middle-income compared with high-income countries. CONCLUSIONS: Poor patient outcomes are common after inpatient surgery. Global initiatives to increase access to surgical treatments should also address the need for safe perioperative care. STUDY REGISTRATION: ISRCTN5181700
Layered structure and complex mechanochemistry underlie strength and versatility in a bacterial adhesive
While designing synthetic adhesives that perform in aqueous environments has proven challenging, microorganisms commonly produce bioadhesives that efficiently attach to a variety of substrates, including wet surfaces. The aquatic bacterium Caulobacter crescentus uses a discrete polysaccharide complex, the holdfast, to strongly attach to surfaces and resist flow. The holdfast is extremely versatile and has impressive adhesive strength. Here, we used atomic force microscopy in conjunction with superresolution microscopy and enzymatic assays to unravel the complex structure of the holdfast and to characterize its chemical constituents and their role in adhesion. Our data support a model whereby the holdfast is a heterogeneous material organized as two layers: a stiffer nanoscopic core layer wrapped into a sparse, far-reaching, flexible brush layer. Moreover, we found that the elastic response of the holdfast evolves after surface contact from initially heterogeneous to more homogeneous. From a composition point of view, besides N-acetyl-d-glucosamine (NAG), the only component that had been identified to date, our data show that the holdfast contains peptides and DNA. We hypothesize that, while polypeptides are the most important components for adhesive force, the presence of DNA mainly impacts the brush layer and the strength of initial adhesion, with NAG playing a primarily structural role within the core. The unanticipated complexity of both the structure and composition of the holdfast likely underlies its versatility as a wet adhesive and its distinctive strength. Continued improvements in understanding of the mechanochemistry of this bioadhesive could provide new insights into how bacteria attach to surfaces and could inform the development of new adhesives
Physiochemical Properties of <i>Caulobacter crescentus</i> Holdfast: A Localized Bacterial Adhesive
To
colonize surfaces, the bacterium <i>Caulobacter crescentus</i> employs a polar polysaccharide, the holdfast, located at the end
of a thin, long stalk protruding from the cell body. Unlike many other
bacteria which adhere through an extended extracellular polymeric
network, the holdfast footprint area is tens of thousands times smaller
than that of the total bacterium cross-sectional surface, making for
some very demanding adhesion requirements. At present, the mechanism
of holdfast adhesion remains poorly understood. We explore it here
along three lines of investigation: (a) the impact of environmental
conditions on holdfast binding affinity, (b) adhesion kinetics by
dynamic force spectroscopy, and (c) kinetic modeling of the attachment
process to interpret the observed time-dependence of the adhesion
force at short and long time scales. A picture emerged in which discrete
molecular units called adhesins are responsible for initial holdfast
adhesion, by acting in a cooperative manner