The antigen 43 structure reveals a molecular velcro-like mechanism of autotransporter-mediated bacterial clumping

Aggregation and biofilm formation are critical mechanisms for bacterial resistance to host immune factors and antibiotics. Autotransporter (AT) proteins, which represent the largest group of outer-membrane and secreted proteins in Gram-negative bacteria, contribute significantly to these phenotypes. Despite their abundance and role in bacterial pathogenesis, most AT proteins have not been structurally characterized, and there is a paucity of detailed information with regard to their mode of action. Here we report the structure–function relationships of Antigen 43 (Ag43a), a prototypic self-associating AT protein from uropathogenic Escherichia coli. The functional domain of Ag43a displays a twisted L-shaped β-helical structure firmly stabilized by a 3D hydrogen-bonded scaffold. Notably, the distinctive Ag43a L shape facilitates self-association and cell aggregation. Combining all our data, we define a molecular “Velcro-like” mechanism of AT-mediated bacterial clumping, which can be tailored to fit different bacterial lifestyles such as the formation of biofilms.

Physical mechanism of the compressive response of F-actin networks : significance of crosslinker unbinding events

A model of crosslinker unbinding is implemented in a highly coarsegrained granular model of F-actin cytoskeleton. We employ this specific granular model to study the mechanisms of the compressive responses of F-actin networks. It is found that the compressive response of F-actin cytoskeleton has dependency on the strain rate. The evolution of deformation energy in the network indicates that crosslinker unbinding events can induce the remodelling of F-actin cytoskeleton in response to external loadings. The internal stress in F-actin cytoskeleton can efficiently dissipate with the help of crosslinker unbinding, which could lead to the spontaneous relaxation of living cells.

A biomarker panel to discriminate between systemic inflammatory response syndrome and sepsis and sepsis severity

Introduction: In this study, we report on initial efforts to discover putative biomarkers for differential diagnosis of a systemic inflammatory response syndrome (SIRS) versus sepsis; and different stages of sepsis. In addition, we also investigated whether there are proteins that can discriminate between patients who survived sepsis from those who did not. Materials and Methods: Our study group consisted of 16 patients, of which 6 died and 10 survived. We daily measured 28 plasma proteins, for the whole stay of the patients in the ICU. Results: We observed that metalloproteinases and sE-selectin play a role in the distinction between SIRS and sepsis, and that IL-1, IP-10, sTNF-R2 and sFas appear to be indicative for the progression from sepsis to septic shock. A combined measurement of MMP-3, -10, IL-1, IP-10, sIL-2R, sFas, sTNF-R1, sRAGE, GM-CSF, IL-1 and Eotaxin allows for a good separation of patients that survived from those that died (mortality prediction with a sensitivity of 79% and specificity of 86%). Correlation analysis suggests a novel interaction between IL-1a and IP-10. Conclusion: The marker panel is ready to be verified in a validation study with or without therapeutic intervention.

Teachers facilitating support for young children experiencing parental separation and divorce

This paper reports on a study of Australian early childhood teachers’ pedagogical practices with young children experiencing parental separation and divorce. Twenty-one semi-structured interviews and a focus group were conducted to explore the actions of teachers to support young children experiencing parental separation and divorce. A grounded theory approach was used to analyse data. Teachers reported actions that were focussed on constructing emotional, behavioural, and academic support for young children, as well as forming partnerships with parents, school personnel, and community members to assist. Results are discussed in terms of the implications for professional practice.

The decision-making processes of early childhood teachers when working with children experiencing parental separation and divorce

In this study, the pedagogical decision-making processes of 21 Australian early childhood teachers working with children experiencing parental separation and divorce were examined. Transcripts from interviews and a focus group with teachers were analysed using grounded theory methodology. The findings showed that as teachers interacted with young children experiencing parental separation and divorce, they reported using strategic, reflexive pedagogical decision-making processes. These processes comprised five stages: (1) teachers constructing their knowledge; (2) teachers thinking about their knowledge; (3) teachers using decision-making schemas; (4) teachers taking action, and; (5) teachers monitoring action and evaluating. This understanding of teachers’ reflexive pedagogical decision-making is useful for identifying how teachers and educational leaders can support children experiencing parental separation and divorce or other life challenges.

Mechanism of kaolinite sheets curling via the intercalation and delamination process

Kaolinite naturally occurs in the plate form for the interlayer hydrogen bond and the distortion and adaption of tetrahedron and octahedron. But kaolinite sheets can be exfoliated to nanoscrolls artificially in laboratory through multiple-step displacement intercalation. The driving force for kaolinite sheet to be curled nanoscroll originates from the size discrepancy of Si–O tetrahedron and Al–O octahedron. The displacement intercalation promoted the platy kaolinite sheets spontaneously to be scrolled by eliminating the interlayer hydrogen bond and atomic interaction. Kaolinite nanoscrolls are hollow tubes with outer face of tetrahedral sheet and inner face of octahedral sheet. Based on the theoretical calculation it is firstly reported that the minimum interior diameter for a single kaolinite sheet to be scrolled is about 9.08 nm, and the optimal 24.30 nm, the maximum 100 nm, which is verified by the observation of scanning electron microscope and transmission electron microscope. The different adaption types and discrepancy degree between tetrahedron and octahedron generate various curling forces in different directions. The nanoscroll axes prefer the directions as [100], [1 �10], [110], [3 �10], and the relative curling force are as follows, [3 �10] > [100] = [1�10] > [110].

Conformational Spread as a Mechanism for Cooperativity in the Bacterial Flagellar Switch

The bacterial flagellar switch that controls the direction of flagellar rotation during Chemotaxis has a highly cooperative response. This has previously been understood in terms of the classic two-state, concerted model of allosteric regulation. Here, we used high-resolution optical microscopy to observe switching of single motors and uncover the stochastic multistate nature of the switch. Our observations are in detailed quantitative agreement with a recent general model of allosteric cooperativity that exhibits conformational spread-the stochastic growth and shrinkage of domains of adjacent subunits sharing a particular conformational state. We expect that conformational spread will be important in explaining cooperativity in other large signaling complexes.

Large-volume methacrylate monolith for plasmid purification : process engineering approach to synthesis and application

The extent of exothermicity associated with the construction of large-volume methacrylate monolithic columns has somewhat obstructed the realisation of large-scale rapid biomolecule purification especially for plasmid-based products which have proven to herald future trends in biotechnology. A novel synthesis technique via a heat expulsion mechanism was employed to prepare a 40 mL methacrylate monolith with a homogeneous radial pore structure along its thickness. Radial temperature gradient was recorded to be only 1.8 °C. Maximum radial temperature recorded at the centre of the monolith was 62.3 °C, which was only 2.3 °C higher than the actual polymerisation temperature. Pore characterisation of the monolithic polymer showed unimodal pore size distributions at different radial positions with an identical modal pore size of 400 nm. Chromatographic characterisation of the polymer after functionalisation with amino groups displayed a persistent dynamic binding capacity of 15.5 mg of plasmid DNA/mL. The maximum pressure drop recorded was only 0.12 MPa at a flow rate of 10 mL/min. The polymer demonstrated rapid separation ability by fractionating Escherichia coli DH5α-pUC19 clarified lysate in only 3 min after loading. The plasmid sample collected after the fast purification process was tested to be a homogeneous supercoiled plasmid with DNA electrophoresis and restriction analysis.

Preparation of macroporous methacrylate monolithic material with convective flow properties for bioseparation : Investigating the kinetics of pore formation and hydrodynamic performance

The preparation of macroporous methacrylate monolithic material with controlled pore structures can be carried out in an unstirred mould through careful and precise control of the polymerisation kinetics and parameters. Contemporary synthesis conditions of methacrylate monolithic polymers are based on existing polymerisation schemes without an in-depth understanding of the dynamics of pore structure and formation. This leads to poor performance in polymer usage thereby affecting final product recovery and purity, retention time, productivity and process economics. The unique porosity of methacrylate monolithic polymer which propels its usage in many industrial applications can be controlled easily during its preparation. Control of the kinetics of the overall process through changes in reaction time, temperature and overall composition such as cross-linker and initiator contents allow the fine tuning of the macroporous structure and provide an understanding of the mechanism of pore formation within the unstirred mould. The significant effect of temperature of the reaction kinetics serves as an effectual means to control and optimise the pore structure and allows the preparation of polymers with different pore size distributions from the same composition of the polymerisation mixture. Increasing the concentration of the cross-linking monomer affects the composition of the final monoliths and also decreases the average pore size as a result of pre-mature formation of highly cross-linked globules with a reduced propensity to coalesce. The choice and concentration of porogen solvent is also imperative. Different porogens and porogen mixtures present different pore structure output. Example, larger pores are obtained in a poor solvent due to early phase separation.

Binding mechanism of affinity ligands for purification of plasmid DNA

Plasmid DNA for therapeutic and vaccination purposes must be highly purified. The high selectivity of affinity chromatography makes it ideal for the isolation of pDNA from complex biological feed stocks. Affinity chromatography makes use of the biological function and/or individual chemical structure of the interacting molecules. However, the success of any affinity purification protocol is dependent on the availability of suitable ligands. In this study, surface plasmon resonance (SPR) based Biacore system has been employed for the detection and quantification of the binding between lac operon (lacO) sequence contained in a pDNA and synthetic peptides based on the DNA-binding domain of the lac repressor protein, lad. The equilibrium dissociation constant (K D) and association and dissociation rate constants (ka, kd) for the interaction between plasmid DNA and designed peptides were determined.

Enhancing methacrylate-monolith-based downstream processes to champion plasmid DNA production

Increasing numbers of preclinical and clinical studies are utilizing pDNA (plasmid DNA) as the vector. In addition, there has been a growing trend towards larger and larger doses of pDNA utilized in human trials. The growing demand on pDNA manufacture leads to pressure to make more in less time. A key intervention has been the use of monoliths as stationary phases in liquid chromatography. Monolithic stationary phases offer fast separation to pDNA owing to their large pore size, making pDNA in the size range from 100 nm to over 300 nm easily accessible. However, the convective transport mechanism of monoliths does not guarantee plasmid purity. The recovery of pure pDNA hinges on a proper balance in the properties of the adsorbent phase, the mobile phase and the feedstock. The effects of pH and ionic strength of binding buffer, temperature of feedstock, active group density and the pore size of the stationary phase were considered as avenues to improve the recovery and purity of pDNA using a methacrylate-based monolithic adsorbent and Escherichia coli DH5α-pUC19 clarified lysate as feedstock. pDNA recovery was found to be critically dependent on the pH and ionic strength of the mobile phase. Up to a maximum of approx. 92% recovery was obtained under optimum conditions of pH and ionic strength. Increasing the feedstock temperature to 80°C increased the purity of pDNA owing to the extra thermal stability associated with pDNA over contaminants such as proteins. Results from toxicological studies of the plasmid samples using endotoxin standard (E. coli 0.55:B5 lipopolysaccharide) show that endotoxin level decreases with increasing salt concentration. It was obvious that large quantities of pure pDNA can be obtained with minimal extra effort simply by optimizing process parameters and conditions for pDNA purification.

A recurrent network in the lateral amygdala: A mechanism for temporal coincidence detection

The dorsal lateral amygdala (LAd) is a vital nucleus for the formation of associations between aversive unconditioned stimuli (US) and neutral stimuli, such as auditory tones, which can become conditioned (CS) to the US through temporal pairing. Important aspects of CS-US associations are believed to occur within the LAd, however relatively little is known about the temporal behavior of local LAd networks. Information about the CS and US enters the LA via a rapid and direct thalamic input and a longer latency cortical path...

Cooperative choice and its framing effect under threshold uncertainty in a provision point mechanism

This paper explores how threshold uncertainty affects cooperative behaviors in the provision of public goods and the prevention of public bads. The following facts motivate our study. First, environmental (resource) problems are either framed as public bads prevention or public goods provision. Second, the occurrence of these problems is characterized by thresholds that are interchangeably represented as "nonconvexity," "bifurcation," "bi-stability," or "catastrophes." Third, the threshold location is mostly unknown. We employ a provision point mechanism with threshold uncertainty and analyze the responses of cooperative behaviors to uncertainty and to the framing for each type of social preferences categorized by a value orientation test. We find that aggregate framing effects are negligible, although the response to the frame is the opposite depending on the type of social preferences. "Cooperative" subjects become more cooperative in negative frames than in positive frames, whereas "individualistic" subjects are less cooperative in negative frames than in positive ones. This finding implies that the insignificance of aggregate framing effects arises from behavioral asymmetry. We also find that the percentage of cooperative choices non-monotonically varies with the degree of threshold uncertainty, irrespective of framing and value orientation. Specifically, the degree of cooperation is highest at intermediate levels of threshold uncertainty and decreases as the uncertainty becomes sufficiently large.