Soluble NSF attachment protein receptor molecular mimicry by a Legionella pneumophila Dot/Icm effector

Upon infection, Legionella pneumophila uses the Dot/Icm type IV secretion system to translocate effector proteins from the Legionella-containing vacuole (LCV) into the host cell cytoplasm. The effectors target a wide array of host cellular processes that aid LCV biogenesis, including the manipulation of membrane trafficking. In this study, we used a hidden Markov model screen to identify two novel, non-eukaryotic soluble NSF attachment protein receptor (SNARE) homologs: the bacterial Legionella SNARE effector A (LseA) and viral SNARE homolog A proteins. We characterized LseA as a Dot/Icm effector of L. pneumophila, which has close homology to the Qc-SNARE subfamily. The lseA gene was present in multiple sequenced L. pneumophila strains including Corby and was well distributed among L. pneumophila clinical and environmental isolates. Employing a variety of biochemical, cell biological and microbiological techniques, we found that farnesylated LseA localized to membranes associated with the Golgi complex in mammalian cells and LseA interacted with a subset of Qa-, Qb- and R-SNAREs in host cells. Our results suggested that LseA acts as a SNARE protein and has the potential to regulate or mediate membrane fusion events in Golgi-associated pathways.

A neuronal topography of visual and acoustic fear conditioning within the amygdala

The lateral amygdala (LA) receives information from auditory and visual sensory modalities, and uses this information to encode lasting memories that predict threat. One unresolved question about the amygdala is how multiple memories, derived from different sensory modalities, are organized at the level of neuronal ensembles. We previously showed that fear conditioning using an auditory conditioned stimulus (CS) was spatially allocated to a stable topography of neurons within the dorsolateral amygdala (LAd) (Bergstrom et al, 2011). Here, we asked how fear conditioning using a visual CS is topographically organized within the amygdala. To induce a lasting fear memory trace we paired either an auditory (2 khz, 55 dB, 20 s) or visual (1 Hz, 0.5 s on/0.5 s off, 35 lux, 20 s) CS with a mild foot shock unconditioned stimulus (0.6 mA, 0.5 s). To detect learning-induced plasticity in amygdala neurons, we used immunohistochemistry with an antibody for phosphorylated mitogen-activated protein kinase (pMAPK). Using a principal components analysis-based approach to extract and visualize spatial patterns, we uncovered two unique spatial patterns of activated neurons in the LA that were associated with auditory and visual fear conditioning. The first spatial pattern was specific to auditory cued fear conditioning and consisted of activated neurons topographically organized throughout the LAd and ventrolateral nuclei (LAvl) of the LA. The second spatial pattern overlapped for auditory and visual fear conditioning and was comprised of activated neurons located mainly within the LAvl. Overall, the density of pMAPK labeled cells throughout the LA was greatest in the auditory CS group, even though freezing in response to the visual and auditory CS was equivalent. There were no differences detected in the number of pMAPK activated neurons within the basal amygdala nuclei. Together, these results provide the first basic knowledge about the organizational structure of two different fear engrams within the amygdala and suggest they are dissociable at the level of neuronal ensembles within the LA

Neurobiological correlates of encoding strength of Pavlovian fear conditioned memory

Emotionally significant memories, especially those induced in conjunction with physical and mental trauma, are frequently retained for an individual’s lifetime. How these memories are organized and encoded within neural networks is a fundamental question. The lateral amygdala (LA) is a key nucleus for acquisition and maintenance of associative emotional memories. We used Pavlovian fear conditioning to study how ‘weaker’ and ‘stronger’ memories are encoded in neural networks of the LA. In Pavlovian fear conditioning a neutral stimulus, in this case a tone, is temporally paired with an aversive unconditioned stimulus (US), such as a foot shock. The previously neutral stimulus becomes a conditioned stimulus (CS) capable of eliciting defensive responses. We used time spent freezing when the CS is presented in a neutral context as a dependent variable measure of memory ‘strength’.

Green-fluorescent protein facilitates rapid in vivo detection of genetically transformed plant cells

Early detection of plant transformation events is necessary for the rapid establishment and optimization of plant transformation protocols. We have assessed modified versions of the green fluorescent protein (GFP) from Aequorea victoria as early reporters of plant transformation using a dissecting fluorescence microscope with appropriate filters. Gfp-expressing cells from four different plant species (sugarcane, maize, lettuce, and tobacco) were readily distinguished, following either Agrobacterium-mediated or particle bombardment-mediated transformation. The identification of gfp-expressing sugarcane cells allowed for the elimination of a high proportion of non-expressing explants and also enabled visual selection of dividing transgenic cells, an early step in the generation of transgenic organisms. The recovery of transgenic cell clusters was streamlined by the ability to visualize gfp-expressing tissues in vitro.

Experimental studies of cold-formed steel hollow section columns at elevated temperatures

This paper reports the details of an experimental study of cold-formed steel hollow section columns at ambient and elevated temperatures. In this study the global buckling behaviour of cold-formed Square Hollow Section (SHS) slender columns under axial compression was investigated at various uniform elevated temperatures up to 700℃. The results of these column tests are reported in this paper, which include the buckling/failure modes at elevated temperatures, and ultimate load versus temperature curves. Finite element models of tested columns were also developed and their behaviour and ultimate capacities at ambient and elevated temperatures were studied. Fire design rules given in European and American standards including the Direct Strength Method (DSM) based design rules were used to predict the ultimate capacities of tested columns at elevated temperatures. Elevated temperature mechanical properties and stress-strain models given in European steel design standards and past researches were used with design rules and finite element models to investigate their effects on SHS column capacities. Comparisons of column capacities from tests and finite element analyses with those predicted by current design rules were used to determine the accuracy of currently available column design rules in predicting the capacities of SHS columns at elevated temperatures and the need to use appropriate elevated temperature material stress-strain models. This paper presents the important findings derived from the comparisons of these column capacities.

Web crippling tests of cold-formed steel channels under two flange load cases

Cold-formed steel members have many advantages over hot-rolled steel members. However, they are susceptible to various buckling modes at stresses below the yield stress of the member because of their relatively high width-to-thickness ratio. Web crippling is a form of localized failure mode that can occur when the members are subjected to transverse high concentrated loadings and/or reactions. The four common loading conditions are the end-one-flange (EOF), interior-one-flange (IOF), end-two-flange (ETF) and interior-two-flange (ITF) loadings. Recently a test method has been proposed by AISI to obtain the web crippling capacities under these four loading conditions. Using this test method 42 tests were conducted in this research to investigate the web crippling behaviour and strengths of unlipped channels with stocky webs under ETF and ITF cases. DuraGal sections having a nominal yield stress of 450 MPa were tested with different web slenderness and bearing lengths. The flanges of these channel sections were not fastened to the supports. In this research the suitability of the currently available design rules for unlipped channels subject to web crippling was investigated, and suitable modifications were proposed where necessary. In addition to this, a new design rule was proposed based on the direct strength method to predict the web crippling capacities of tested beams. This paper presents the details of this experimental study and the results.

Local buckling studies of cold-formed steel compression members at elevated temperatures

Cold-formed steel members have been widely used in residential and commercial buildings as primary load bearing structural elements. They are often made of thin steel sheets and hence they are more susceptible to local buckling. The buckling behaviour of cold-formed steel compression members under fire conditions is not fully investigated yet and hence there is a lack of knowledge on the fire performance of cold-formed steel compression members. Current cold-formed steel design standards do not provide adequate design guidelines for the fire design of cold-formed steel compression members. Therefore a research project based on extensive experimental and numerical studies was undertaken to investigate the local buckling behaviour of light gauge cold-formed steel compression members under simulated fire conditions. First a series of 91 local buckling tests was conducted at ambient and uniform elevated temperatures up to 700oC on cold-formed lipped and unlipped channels. Suitable finite element models were then developed to simulate the behaviour of tested columns and were validated using test results. All the ultimate load capacity results for local buckling were compared with the predictions from the available design rules based on AS/NZS 4600, BS 5950 Part 5, Eurocode 3 Parts 1.2 and 1.3 and the direct strength method (DSM), based on which suitable recommendations have been made for the fire design of cold-formed steel compression members subject to local buckling at uniform elevated temperatures.

Lipid-modified azurin of Neisseria meningitidis is a copper protein localized on the outer membrane surface and not regulated by FNR

The laz gene of Neisseria meningitidis is predicted to encode a lipid-modified azurin (Laz). Laz is very similar to azurin, a periplasmic protein, which belongs to the copper-containing proteins in the cupredoxin superfamily. In other bacteria, azurin is an electron donor to nitrite reductase, an important enzyme in the denitrifying process. It is not known whether Laz could function as an electron transfer protein in this important pathogen. Laz protein was heterologously expressed in Escherichia coli and purified. Electrospray mass spectrometry indicated that the Laz protein contains one copper ion. Laz was shown to be redox-active in the presence of its redox center copper ion. When oxidized, Laz exhibits an intense blue colour and absorbs visible light around 626 nm. The absorption is lost when exposed to diethyldithiocarbamate, a copper chelating agent. Polyclonal antibodies were raised against purified Laz for detecting expression of Laz under different growth conditions and to determine the orientation of Laz on the outer membrane. The expression of Laz under microaerobic and microaerobic denitrifying conditions was slightly higher than that under aerobic conditions. However, the expression of Laz was similar between the wild type strain and an fnr mutant, suggesting that Fumarate/Nitrate reduction regulator (FNR) does not regulate the expression of Laz despite the presence of a partial FNR box upstream of the laz gene. We propose that some Laz protein is exposed on the outer membrane surface of N. meningitidis as the αLaz antibodies can increase killing by complement in a capsule deficient N. meningitidis strain, in a dose-dependent fashion.

Cold water immersion: Practices, trends and avenues of effect

Cold water immersion and ice baths are popular methods of recovery used by athletes. From the simple wheelie bin with water and ice, to the inflatable baths with complex water cooling units to recovery sessions in the ocean, the practice of cold water immersion is wide and varied. Research into cold water immersion was conducted as early as 1963 when Clarke1 examined the influence of cold water on performance recovery after a sustained handgrip exercise. Research has been conducted to understand how cold water immersion might affect the body’s physiological systems and how factors such as water temperature and the duration of immersion might enhance recovery after training and/or competition. Despite this research activity, how are we to know if research is being put into practice? In more serious situations, where guidelines and policies need to be standardised for the safe use of a product, one would expect that there is a straight forward follow-on from research into practice. Although cold water immersion may not need the rigor of testing compared to drug treatments, for example, the decision on whether to use cold water immersion in specific situations (e.g. after training or competition) may rest with one or two of the staff associated with the athlete/team. Therefore, it would be expected that these staff are well-informed on the current literature regarding cold water immersion.

The structural basis of DNA binding by the single-stranded DNA-binding protein from Sulfolobus solfataricus

Canonical single-stranded DNA-binding proteins (SSBs) from the oligosaccharide/oligonucleotide-binding (OB) domain family are present in all known organisms and are critical for DNA replication, recombination and repair. The SSB from the hyperthermophilic crenarchaeote Sulfolobus solfataricus (SsoSSB) has a ‘simple’ domain organization consisting of a single DNA-binding OB fold coupled to a flexible C-terminal tail, in contrast with other SSBs in this family that incorporate up to four OB domains. Despite the large differences in the domain organization within the SSB family, the structure of the OB domain is remarkably similar all cellular life forms. However, there are significant differences in the molecular mechanism of ssDNA binding. We have determined the structure of the SsoSSB OB domain bound to ssDNA by NMR spectroscopy. We reveal that ssDNA recognition is modulated by base-stacking of three key aromatic residues, in contrast with the OB domains of human RPA and the recently discovered human homologue of SsoSSB, hSSB1. We also demonstrate that SsoSSB binds ssDNA with a footprint of five bases and with a defined binding polarity. These data elucidate the structural basis of DNA binding and shed light on the molecular mechanism by which these ‘simple’ SSBs interact with ssDNA.

Comparative durability study of CFRP strengthened tubular steel members under cold weather

In strengthening systems, the CFRP (Carbon Fibre Reinforced Polymer) materials typically have excellent resistance against environmental conditions; however, the performance of adhesives between CFRP and steel is generally affected by various environmental conditions such as marine environment, cold and hot weather. This paper presents the comparative durability study of CFRP strengthened tubular steel structures by using two different adhesives such as MBrace saturant and Araldite K630 under four-point bending. The program consisted of testing twelve CFRP strengthened specimens having treated with epoxy based adhesion promoter, untreated surface and one unstrengthened specimen and conditioned under cold weather for 3 and 6 months to determine the environmental durability. The beams were then loaded to failure in quasi-static manner under four-point bending. The structural responses of CFRP strengthened tubular steel beams were compared in terms of failure load, stiffness and modes of failure. The research findings show that the cold weather immersion had adversely affected the durability of CFRP strengthened steel members. Design factor is also proposed to address the short-terms durability performance under cold weather.

Effects of cold water immersion and active recovery on hemodynamics and recovery of muscle strength following resistance exercise

Cold water immersion (CWI) and active recovery (ACT) are frequently used as post-exercise recovery strategies. However, the physiological effects of CWI and ACT after resistance exercise are not well characterized. We examined the effects of CWI and ACT on cardiac output (Q), muscle oxygenation (SmO2) and blood volume (tHb), muscle temperature (Tmuscle ) and isometric strength after resistance exercise. On separate days, 10 men performed resistance exercise, followed by 10 min CWI at 10°C or 10 min ACT (low-intensity cycling). Q (7.9±2.7 l) and Tmuscle (2.2±0.8ºC) increased, whereas SmO2 (-21.5±8.8%) and tHb (-10.1±7.7 μM) decreased after exercise (p<0.05). During CWI, Q ̇(-1.1±0.7 l) and Tmuscle (-6.6±5.3ºC) decreased, while tHb (121±77 μM) increased (p<0.05). In the hour after CWI, Q ̇and Tmuscle remained low, while tHb also decreased (p<0.05). By contrast, during ACT, Q ̇(3.9±2.3 l), Tmuscle (2.2±0.5ºC), SmO2 (17.1±5.7%) and tHb (91±66 μM) all increased (p<0.05). In the hour after ACT, Tmuscle and tHb remained high (p<0.05). Peak isometric strength during 10 s maximum voluntary contractions (MVCs) did not change significantly after CWI, whereas it decreased after ACT (-30 to -45 Nm; p<0.05). Muscle deoxygenation time during MVCs increased after ACT (p<0.05), but not after CWI. Muscle reoxygenation time after MVCs tended to increase after CWI (p=0.052). These findings suggest firstly that hemodynamics and muscle temperature after resistance exercise are dependent on ambient temperature and metabolic demands with skeletal muscle, and secondly, that recovery of strength after resistance exercise is independent of changes in hemodynamics and muscle temperature.

Fire design rules for load bearing cold-formed steel frame walls exposed to realistic design fire curves

Light gauge Steel Frame (LSF) walls are extensively used in the building industry due to the many advantages they provide over other wall systems. Although LSF walls have been used widely, fire design of LSF walls is based on approximate prescriptive methods based on limited fire tests. Also these fire tests were conducted using the standard fire curve [1] and the applicability of available design rules to realistic design fire curves has not been verified. This paper investigates the accuracy of existing fire design rules in the current cold-formed steel standards and the modifications proposed by previous researchers. Of these the recently developed design rules by Gunalan and Mahendran [2] based on Eurocode 3 Part 1.3 [3] and AS/NZS 4600 [4] for standard fire exposure [1] were investigated in detail to determine their applicability to predict the axial compression strengths and fire resistance ratings of LSF walls exposed to realistic design fire curves. This paper also presents the fire performance results of LSF walls exposed to a range of realistic fire curves obtained using a finite element analysis based parametric study. The results from the parametric study were used to develop a simplified design method based on the critical hot flange temperature to predict the fire resistance ratings of LSF walls exposed to realistic fire curves. Finally, the stud failure times (fire resistance rating) obtained from the fire design rules and the simplified design method were compared with parametric study results for LSF walls lined with single and double plasterboards, and externally insulated with rock fibres under realistic fire curves.

Evaluation of protein bait laced with various insecticides on the Queensland fruit fly (Diptera: Tephritidae): Attraction, feeding, mortality and bait persistence

The use of malathion in fruit fly protein bait sprays has raised serious concerns due to its adverse effects on non-target organisms. This has necessitated the evaluation of novel reduced-risk compounds. This study evaluated the effects of spinosad, fipronil, malathion and chlorpyrifos mixed with fruit fly protein bait (Mauri Pinnacle protein®) on attraction, feeding and mortality of the Queensland fruit fly, Bactrocera tryoni (Froggatt). The effects of outdoor weathering of these mixtures on fly mortality were also determined. In field-cage experiment, protein-starved flies showed the same level of attraction to baits containing spinosad, fipronil, malathion, chlorpyrifos and protein alone used as control. Female protein-starved flies were deterred from feeding on baits containing malathion and chlorpyrifos compared to baits containing spinosad, fipronil and protein alone. Baits containing malathion and chlorpyrifos caused higher fly mortality and rapid fly knock down than spinosad and fipronil. However, spinosad acted slowly and caused an increase in fly mortality over time, causing up to 90% fly mortality after 72-h. Baits containing malathion and chlorpyrifos, applied on citrus leaves and weathered outdoors, had longer residual effectiveness in killing flies than spinosad and fipronil. Residual effectiveness of the spinosad bait mixture waned significantly after 3 days of outdoor weathering. Results suggest that spinosad and fipronil can be potential alternatives for malathion in protein bait sprays.

Numerical modelling and design of unlined cold-formed steel wall frames

Cold-formed steel wall frame systems using lipped or unlipped C-sections and gypsum plasterboard lining are commonly utilised in the construction of both the load bearing and non-load bearing walls in the residential, commercial and industrial buildings. However, the structural behaviour of unlined and lined stud wall frames is not well understood and adequate design rules are not available. A detailed research program was therefore undertaken to investigate the behaviour of stud wall frame systems. As the first step in this research, the problem relating to the degree of end fixity of stud was investigated. The studs are usually connected to the top and bottom tracks and the degree of end fixity provided by these tracks is not adequately addressed by the design codes. A finite element model of unlined frames was therefore developed, and validated using full scale experimental results. It was then used in a detailed parametric study to develop appropriate design rules for unlined wall frames. This study has shown that by using appropriate effective length factors, the ultimate load and failure modes of the unlined studs can be accurately predicted using the provisions of Australian or American cold-formed steel structures design codes. This paper presents the details of the finite element analyses, the results and recommended design rules for unlined wall frames.