Surveillance of central line associated bloodstream infection (CLABSI) - comparison of current (CDC/NHSN) and modified criteria: A prospective study.

Background and Aims: There is a huge load of central line-associated bloodstream infection (CLABSI) being reported in developing countries, with increased mortality and healthcare costs. Effective surveillance is a must to reduce the incidence of CLABSI. The current criteria (Centre for Disease Control and Prevention/National Healthcare Safety Network [CDC/NHSN]) for CLABSI surveillance have their own shortcomings. For diagnosing CLABSI, current CDC/NHSN CLABSI surveillance criteria are laborious and time consuming with low predictive power. Hence, modified criteria have been postulated, which are simple and implementable at resource-constrained setups. The primary objective was to compare modified criteria with CDC criteria. The secondary objective was to determine the prevalence of CRBSI. Material and Methods: A total of 98 patients with central line in situ or having the central venous line removed ≤24 hrs prior to the date of the event were enrolled. Paired blood cultures were obtained and results were analyzed using differential time to positivity. Results: The incidence of CLBSI was 8.16% and the device utilization rate was 11.6%. The negative predictive value of both the surveillance criteria was found to be excellent and comparable (96.2% for modified criteria and 97.1% for CDC criteria), therefore both can be used for screening purposes. AUC for current CDC/NHSN criteria was better than modified criteria (0.76 versus 0.66, P < 0.0001), suggesting it to be a better criterion for surveillance of CLABSI. Conclusion: Modified criteria were not superior to CDC/NHSN criteria for surveillance. Thus, there is a scope of improving the modified criteria for the purpose of surveillance. CLBSI load was higher; CLABSI bundle for prevention is thus highly recommended.

Complex pH-Dependent Interactions between Weak Polyelectrolyte Block Copolymer Micelles and Molecular Fluorophores.

Amphiphilic block copolymers with weak polyelectrolyte blocks can assemble stimulus-responsive nanostructures and interfaces. Applications of these materials in drug delivery, biomimetics, and sensing largely rely on the well-understood swelling of polyelectrolyte chains upon deprotonation, often induced by changes in pH or ionic strength. This deprotonation can also tune interfacial interactions between the polyelectrolyte blocks and surrounding solution, an effect which is less studied than morphological swelling of polyelectrolytes but can be just as critical for intended function. Here, we investigate whether the pH-driven morphological response of polyelectrolyte-bearing nanostructures also affects the interactions of these nanostructures with molecules in solution, using micelles of a short-chain polybutadiene-block-poly(acrylic acid) (pBd-pAA) as a model system. We introduce a Förster resonance energy transfer (FRET) approach to probe interactions between micelles and fluorescent molecular solutes as a function of solution pH. As expected, the pAA corona of these pBd-pAA micelles increases in thickness monotonically as a function of pH. However, FRET efficiency, which provides a metric of the spatial proximity of fluorescently labeled micelles and freely diffusing fluorophores, exhibits complex nonmonotonic behavior as a function of pH, indicating that the average separation of micelles and acceptor fluorophores is not strictly correlated with micelle swelling. Dialysis experiments quantify the affinity of fluorophores for micelles as a function of pH, confirming that changes in FRET are driven almost entirely by the pH-dependent affinity of the pAA block for the investigated molecular fluorophores, not simply by a shape change of the pAA corona. This study provides key insights into the interfacial interactions between weak-polyelectrolyte-bearing nanostructures and molecular solutes, of importance for the development of their stimulus-responsive applications.

High-pressure small-angle X-ray scattering cell for biological solutions and soft materials.

Pressure is a fundamental thermodynamic parameter controlling the behavior of biological macromolecules. Pressure affects protein denaturation, kinetic parameters of enzymes, ligand binding, membrane permeability, ion trans-duction, expression of genetic information, viral infectivity, protein association and aggregation, and chemical processes. In many cases pressure alters the molecular shape. Small-angle X-ray scattering (SAXS) is a primary method to determine the shape and size of macromolecules. However, relatively few SAXS cells described in the literature are suitable for use at high pressures and with biological materials. Described here is a novel high-pressure SAXS sample cell that is suitable for general facility use by prioritization of ease of sample loading, temperature control, mechanical stability and X-ray background minimization. Cell operation at 14 keV is described, providing a q range of 0.01 < q < 0.7 Å-1, pressures of 0-400 MPa and an achievable temperature range of 0-80°C. The high-pressure SAXS cell has recently been commissioned on the ID7A beamline at the Cornell High Energy Synchrotron Source and is available to users on a peer-reviewed proposal basis.

Structural determination of Enzyme-Graphene Nanocomposite Sensor Material.

State-of-the-art ultra-sensitive blood glucose-monitoring biosensors, based on glucose oxidase (GOx) covalently linked to a single layer graphene (SLG), will be a valuable next generation diagnostic tool for personal glycemic level management. We report here our observations of sensor matrix structure obtained using a multi-physics approach towards analysis of small-angle neutron scattering (SANS) on graphene-based biosensor functionalized with GOx under different pH conditions for various hierarchical GOx assemblies within SLG. We developed a methodology to separately extract the average shape of GOx molecules within the hierarchical assemblies. The modeling is able to resolve differences in the average GOx dimer structure and shows that treatment under different pH conditions lead to differences within the GOx at the dimer contact region with SLG. The coupling of different analysis methods and modeling approaches we developed in this study provides a universal approach to obtain detailed structural quantifications, for establishing robust structure-property relationships. This is an essential step to obtain an insight into the structure and function of the GOx-SLG interface for optimizing sensor performance.

The consequences of cavity creation on the folding landscape of a repeat protein depend upon context.

The effect of introducing internal cavities on protein native structure and global stability has been well documented, but the consequences of these packing defects on folding free-energy landscapes have received less attention. We investigated the effects of cavity creation on the folding landscape of the leucine-rich repeat protein pp32 by high-pressure (HP) and urea-dependent NMR and high-pressure small-angle X-ray scattering (HPSAXS). Despite a modest global energetic perturbation, cavity creation in the N-terminal capping motif (N-cap) resulted in very strong deviation from two-state unfolding behavior. In contrast, introduction of a cavity in the most stable, C-terminal half of pp32 led to highly concerted unfolding, presumably because the decrease in stability by the mutations attenuated the N- to C-terminal stability gradient present in WT pp32. Interestingly, enlarging the central cavity of the protein led to the population under pressure of a distinct intermediate in which the N-cap and repeats 1-4 were nearly completely unfolded, while the fifth repeat and the C-terminal capping motif remained fully folded. Thus, despite modest effects on global stability, introducing internal cavities can have starkly distinct repercussions on the conformational landscape of a protein, depending on their structural and energetic context.

Grazing-Angle Neutron Diffraction Study of the Water Distribution in Membrane Hemifusion: From the Lamellar to Rhombohedral Phase.

The water distribution between lipid bilayers is important in understanding the role of the hydration force at different steps of the membrane fusion pathway. In this study, we used grazing-angle neutron diffraction to map out the water distribution in lipid bilayers transiting from a lamellar structure to the hemifusion "stalk" structure in a rhombohedral phase. Under osmotic pressure exerted by different levels of relative humidity, the lipid membrane sample was maintained in equilibrium at different lattices suitable for neutron diffraction. The D2O used to hydrate the lipid membrane sample stood out from the lipid in the reconstructed structure because of its much higher coherent neutron scattering length density. The density map indicates that water dissociated from the headgroup in the lamellar phase. In the rhombohedral phase, water was significantly reduced and was squeezed into pockets around the stalk. This study complements earlier structural studies by grazing-angle X-ray diffraction, which is sensitive to only the parts of the structure with high electron density (such as phosphors). The experiment also demonstrated that the recently developed time-of-flight small-angle neutron scattering beamline at the Spallation Neutron Source is suitable for grazing-angle neutron diffraction to provide the structures of large unit cells on the order of a few nanometers, such as biomembrane structures.

Interaction of the Antimicrobial Peptide Aurein 1.2 and Charged Lipid Bilayer.

Aurein 1.2 is a potent antimicrobial peptide secreted by frog Litoria aurea. As a short membrane-active peptide with only 13 amino acids in sequence, it has been found to be residing on the surface of lipid bilayer and permeabilizing bacterial membranes at high concentration. However, the detail at the molecular level is largely unknown. In this study, we investigated the action of Aurein 1.2 in charged lipid bilayers composed of DMPC/DMPG. Oriented Circular Dichroism results showed that the peptide was on the surface of lipid bilayer regardless of the charged lipid ratio. Only at a very high peptide-to-lipid ratio (~1/10), the peptide became perpendicular to the bilayer, however no pore was detected by neutron in-plane scattering. To further understand how it interacted with charged lipid bilayers, we employed Small Angle Neutron Scattering to probe lipid distribution across bilayer leaflets in lipid vesicles. The results showed that Aurein 1.2 interacted strongly with negatively charged DMPG, causing strong asymmetry in lipid bilayer. At high concentration, while the vesicles were intact, we found additional structure feature on the bilayer. Our study provides a glimpse into how Aurein 1.2 disturbs anionic lipid-containing membranes without pore formation.

Changes in lipid bilayer structure caused by the helix-to-sheet transition of an HIV-1 gp41 fusion peptide derivative.

HIV-1, like other enveloped viruses, undergoes fusion with the cell membrane to infect it. Viral coat proteins are thought to bind the virus to the membrane and actively fuse the viral and cellular membranes together. The actual molecular mechanism of fusion is challenging to visualize, resulting in the use of model systems. Here, the bilayer curvature modifying properties of a synthetic variant of the HIV-1 gp41 fusion peptide with lipid bilayer vesicles composed of a mixture of dimyristoyl phosphatidylcholine (DMPC) and dimyristoyl phosphatidylserine (DMPS) were studied. In 7:3 DMPC:DMPS vesicles made with deuterium-labeled DMPC, the peptide was observed to undergo a concentration-dependent conformational transition between an α-helix and an antiparallel ß-sheet. Through the use of small-angle neutron scattering (SANS) and selective deuterium labeling, it was revealed that conformational transition of the peptide is also accompanied by a transition in the structure of the lipid bilayer. In addition to changes in the distribution of the lipid between the leaflets of the vesicle, the SANS data are consistent with two regions having different thicknesses. Of the two different bilayer structures, the one corresponding to the smaller area fraction, being ∼8% of the vesicle area, is much thicker than the remainder of the vesicle, which suggests that there are regions of localized negative curvature similar to what takes place at the point of contact between two membranes immediately preceding fusion.

Plant-Derived Terpenes: A Feedstock for Specialty Biofuels.

Research toward renewable and sustainable energy has identified specific terpenes capable of supplementing or replacing current petroleum-derived fuels. Despite being naturally produced and stored by many plants, there are few examples of commercial recovery of terpenes from plants because of low yields. Plant terpene biosynthesis is regulated at multiple levels, leading to wide variability in terpene content and chemistry. Advances in the plant molecular toolkit, including annotated genomes, high-throughput omics profiling, and genome editing, have begun to elucidate plant terpene metabolism, and such information is useful for bioengineering metabolic pathways for specific terpenes. We review here the status of terpenes as a specialty biofuel and discuss the potential of plants as a viable agronomic solution for future terpene-derived biofuels.

Neutron Scattering Studies of the Interplay of Amyloid ß Peptide(1-40) and An Anionic Lipid 1,2-dimyristoyl-sn-glycero-3-phosphoglycerol.

The interaction between lipid bilayers and Amyloid ß peptide (Aß) plays a critical role in proliferation of Alzheimer's disease (AD). AD is expected to affect one in every 85 humans by 2050, and therefore, deciphering the interplay of Aß and lipid bilayers at the molecular level is of profound importance. In this work, we applied an array of neutron scattering methods to study the structure and dynamics of Aß(1-40) interacting 1,2-dimyristoyl-sn-glycero-3-phosphoglycerol (DMPG) bilayers. In the structural investigations of lipid bilayer's response to Aß binding, Small Angle Neutron Scattering and Neutron Membrane Diffraction revealed that the Aß anchors firmly to the highly charged DMPG bilayers in the interfacial region between water and hydrocarbon chain, and it doesn't penetrate deeply into the bilayer. This association mode is substantiated by the dynamics studies with high resolution Quasi-Elastic Neutron Scattering experiments, showing that the addition of Aß mainly affects the slower lateral motion of lipid molecules, especially in the fluid phase, but not the faster internal motion. The results revealed that Aß associates with the highly charged membrane in surface with limited impact on the structure, but the altered membrane dynamics could have more influence on other membrane processes.

The Interaction of Melittin with Dimyristoyl Phosphatidylcholine-Dimyristoyl Phosphatidylserine Lipid Bilayer Membranes.

Membrane-active peptides (MAPs), which interact directly with the lipid bilayer of a cell and include toxins and host defense peptides, display lipid composition-dependent activity. Phosphatidylserine (PS) lipids are anionic lipids that are found throughout the cellular membranes of most eukaryotic organisms where they serve as both a functional component and as a precursor to phosphatidylethanolamine lipids. The inner leaflet of the plasma membrane contains more PS than the outer one, and the asymmetry is actively maintained. Here, the impact of the MAP melittin on the structure of lipid bilayer vesicles made of a mixture of phosphatidylcholine and phosphatidylserine was studied. Small-angle neutron scattering of the MAP associated with selectively deuterium-labeled lipid bilayer vesicles revealed how the thickness and lipid composition of phosphatidylserine-containing vesicles change in response to melittin. The peptide thickens the lipid bilayer for concentrations up to P/L=1/500, but membrane thinning results when P/L=1/200. The thickness transition is accompanied by a large change in the distribution of DMPS between the leaflets of the bilayer. The change in composition is driven by electrostatic interactions, while the change in bilayer thickness is driven by changes in the interaction of the peptide with the headgroup region of the lipid bilayer. The results provide new information about lipid-specific interactions that take place in mixed composition lipid bilayer membranes.

Quantification of interaction and topological parameters of polyisoprene star polymers under good solvent conditions.

Mass fractal scaling, reflected in the mass fractal dimension d_{f}, is independently impacted by topology, reflected in the connectivity dimension c, and by tortuosity, reflected in the minimum dimension d_{min}. The mass fractal dimension is related to these other dimensions by d_{f}=cd_{min}. Branched fractal structures have a higher mass fractal dimension compared to linear structures due to a higher c, and extended structures have a lower dimension compared to convoluted self-avoiding and Gaussian walks due to a lower d_{min}. It is found, in this work, that macromolecules in thermodynamic equilibrium display a fixed mass fractal dimension d_{f} under good solvent conditions, regardless of chain topology.  These equilibrium structures accommodate changes in chain topology such as branching c by a decrease in chain tortuosity d_{min}. Symmetric star polymers are used to understand the structure of complex macromolecular topologies. A recently published hybrid Unified scattering function accounts for interarm correlations in symmetric star polymers along with polymer-solvent interaction for chains of arbitrary scaling dimension. Dilute solutions of linear, three-arm and six-arm polyisoprene stars are studied under good solvent conditions in deuterated p-xylene. Reduced chain tortuosity can be viewed as steric straightening of the arms. Steric effects for star topologies are quantified, and it is found that steric straightening of arms is more significant for lower-molecular-weight arms. The observation of constant d_{f} is explained through a modification of Flory-Krigbaum theory for branched polymers.

Small-angle neutron scattering reveals the assembly of alpha-synuclein in lipid membranes.

The aggregation of α-synuclein (asyn), an intrinsically disordered protein (IDP), is a hallmark in Parkinson's disease (PD). We investigated the conformational changes that asyn undergoes in the presence of membrane and membrane mimetics using small-angle neutron scattering (SANS). In solution, asyn is monomeric and unfolded assuming an ensemble of conformers spanning extended and compact conformations. Using the contrast variation technique and SANS, the protein scattering signal in the membrane-protein complexes is selectively highlighted in order to monitor its conformational changes in this environment. We showed that in the presence of phospholipid membranes asyn transitions from a monodisperse state to aggregated structures with sizes ranging from 200 to 900Å coexisting with the monomeric species. Detailed SANS data analysis revealed that asyn aggregates have a hierarchical organization in which clusters of smaller asyn aggregates assemble to form the larger structures. This study provides new insight into the mechanism of asyn aggregation. We propose an aggregation mechanism in which stable asyn aggregates seed the aggregation process and hence the hierarchical assembly of structures. Our findings demonstrate that membrane-induced conformational changes in asyn lead to its heterogeneous aggregation which could be physiologically relevant in its function or in the diseased state.

Determination of the interaction parameter and topological scaling features of symmetric star polymers in dilute solution.

Star polymers provide model architectures to understand the dynamic and rheological effects of chain confinement for a range of complex topological structures like branched polymers, colloids, and micelles. It is important to describe the structure of such macromolecular topologies using small-angle neutron and x-ray scattering to facilitate understanding of their structure-property relationships. Modeling of scattering from linear, Gaussian polymers, such as in the melt, has applied the random phase approximation using the Debye polymer scattering function. The Flory-Huggins interaction parameter can be obtained using neutron scattering by this method. Gaussian scaling no longer applies for more complicated chain topologies or when chains are in good solvents. For symmetric star polymers, chain scaling can differ from ν=0.5(d(f)=2) due to excluded volume, steric interaction between arms, and enhanced density due to branching. Further, correlation between arms in a symmetric star leads to an interference term in the scattering function first described by Benoit for Gaussian chains. In this work, a scattering function is derived which accounts for interarm correlations in symmetric star polymers as well as the polymer-solvent interaction parameter for chains of arbitrary scaling dimension using a hybrid Unified scattering function. The approach is demonstrated for linear, four-arm and eight-arm polyisoprene stars in deuterated p-xylene.

Alamethicin disrupts the cholesterol distribution in dimyristoyl phosphatidylcholine-cholesterol lipid bilayers.

Cell membranes are complex mixtures of lipids, proteins, and other molecules that serve as active, semipermeable barriers between cells, as well as between their internal organelles, and the surrounding medium. Their compositions and structures are tightly regulated to ensure proper function. Cholesterol is a key component in mammalian cellular membranes, where it serves to maintain membrane fluidity and permeability. Here, the interaction of alamethicin, a 20 amino acid residue peptide that creates transmembrane pores in lipid bilayer membranes in a concentration-dependent manner, with bilayer membranes composed of dimyristoyl phosphatidylcholine (DMPC) and cholesterol (Chol) was studied. Small-angle neutron scattering (SANS) data demonstrate that a low concentration of alamethicin (peptide-to-lipid ratio of 1/200) disrupts a lateral inhomogeneity seen in peptide-free DMPC:Chol vesicles, which analysis of the SANS data indicates are Chol-rich and Chol-poor phases having different thicknesses. Alamethicin disrupts this structure, producing laterally homogeneous bilayers that are thinner than either phase of the peptide-free bilayers, and possess a strong asymmetry in the Chol content of the inner and outer bilayer leaflets. The results suggest that a secondary membrane disruption mechanism exists in parallel with the well-understood cytotoxic membrane permeabilization that results when alamethcin forms transmembrane pores. Specifically, the peptide can disrupt laterally organized lipidic structures in cell membranes, as well as significantly perturb the compositions of the inner and outer leaflets of the membrane. The existence of a secondary mechanism of action against cellular membranes for alamethicin raises the possibility that other membrane-active peptides function similarly.