Reduced resting skeletal muscle protein synthesis is rescued by resistance exercise and protein ingestion following short-term energy deficit

The myofibrillar protein synthesis (MPS) response to resistance exercise (REX) and protein ingestion during energy deficit (ED) is unknown. We determined, in young men (n=8) and women (n=7), protein signaling, resting post-absorptive MPS during energy balance [EB: 45 kcal∙(kg FFM∙d)-1] and after 5d of ED [30 kcal∙(kg FFM∙d)-1] as well as MPS while in ED after acute REX in the fasted state and with the ingestion of whey protein (15 and 30 g). Post-absorptive rates of MPS were 27% lower in ED than EB (P<0.001), but REX stimulated MPS to rates equal to EB. Ingestion of 15 and 30 g of protein after REX in ED increased MPS ~16 and ~34% above resting EB, (P<0.02). p70 S6Kthr389 phosphorylation increased above EB only with combined exercise and protein intake (~2-7 fold; P<0.05). In conclusion, short-term ED reduces post-absorptive MPS, however, a bout of REX in ED restores MPS to values observed at rest in EB. The ingestion of protein after REX further increases MPS above resting EB in a dose-dependent manner. We conclude that combining REX with increased protein availability after exercise enhances rates of skeletal muscle protein synthesis during short term ED and could, in the long term, preserve muscle mass.

Flexural–torsional buckling behaviour and design of cold-formed steel compression members at elevated temperatures

Current design rules for the member capacities of cold-formed steel columns are based on the same non-dimensional strength curve for both fixed and pinned-ended columns at ambient temperature. This research has investigated the accuracy of using current ambient temperature design rules in Australia/New Zealand (AS/NZS 4600), American (AISI S100) and European (Eurocode 3 Part 1.3) standards in determining the flexural–torsional buckling capacities of cold-formed steel columns at uniform elevated temperatures using appropriately reduced mechanical properties. It was found that these design rules accurately predicted the member capacities of pin ended lipped channel columns undergoing flexural torsional buckling at elevated temperatures. However, for fixed ended columns with warping fixity undergoing flexural–torsional buckling, the current design rules significantly underestimated the column capacities as they disregard the beneficial effect of warping fixity. This paper has therefore recommended the use of improved design rules developed for ambient temperature conditions to predict the axial compression capacities of fixed ended columns subject to flexural–torsional buckling at elevated temperatures within AS/NZS 4600 and AISI S100 design provisions. The accuracy of the proposed fire design rules was verified using finite element analysis and test results of cold-formed lipped channel columns at elevated temperatures except for low strength steel columns with intermediate slenderness whose behaviour was influenced by the increased nonlinearity in the stress–strain curves at elevated temperatures. Further research is required to include these effects within AS/NZS 4600 and AISI S100 design rules. However, Eurocode 3 Part 1.3 design rules can be used for this purpose by using suitable buckling curves as recommended in this paper.

Fire performance and design of cold-formed steel wall systems

Traditionally, the fire resistance rating of Light gauge steel frame (LSF) wall systems is based on approximate prescriptive methods developed using limited fire tests. These fire tests are conducted using standard fire time-temperature curve given in ISO 834. However, in recent times fire has become a major disaster in buildings due to the increase in fire loads as a result of modern furniture and lightweight construction, which make use of thermoplastics materials, synthetic foams and fabrics. Therefore a detailed research study into the performance of load bearing LSF wall systems under both standard and realistic design fires on one side was undertaken to develop improved fire design rules. This study included both full scale fire tests and numerical studies of eight different LSF wall systems conducted for both the standard fire curve and the recently developed realistic design fire curves. The use of previous fire design rules developed for LSF walls subjected to non-uniform elevated temperature distributions based on AISI design manual and Eurocode 3 Parts 1.2 and 1.3 was investigated first. New simplified fire design rules based on AS/NZS 4600, North American Specification and Eurocode 3 Part 1.3 were then proposed with suitable allowances for the interaction effects of compression and bending actions. The importance of considering thermal bowing, magnified thermal bowing and neutral axis shift in the fire design was also investigated and their effects were included. A spread sheet based design tool was developed based on the new design rules to predict the failure load ratio versus time and temperature curves for varying LSF wall configurations. The accuracy of the proposed design rules was verified using the fire test and finite element analysis results for various wall configurations, steel grades, thicknesses and load ratios under both standard and realistic design fire conditions. A simplified method was also proposed to predict the fire resistance rating of LSF walls based on two sets of equations developed for the load ratio-hot flange temperature and the time-temperature relationships. This paper presents the details of this study on LSF wall systems under fire conditions and the results.

Post-fire mechanical properties of cold-formed steels

Cold-formed steel members have been widely used in residential, industrial and commercial buildings as primary load-bearing and non-load bearing structural elements. These buildings must be properly evaluated after a fire event to assess the nature and extent of structural damage. If the general appearance of the structure is satisfactory after a fire event then the question that has to be answered is how the structural capacity of cold-formed steel members in these buildings has been affected. Elevated temperatures during a fire event affect the structural performance of cold-formed steel members even after cooling down to ambient temperature due to the possible detrimental changes in their mechanical properties. However, the post-fire behaviour of cold-formed steel members has not been investigated in the past and hence there is a need to investigate the post-fire mechanical properties of cold-formed steels. Therefore an experimental study was undertaken at the Queensland University of Technology to understand the residual mechanical properties of cold-formed steels after fire events. Tensile coupon tests were conducted on three different steel grades and thicknesses to obtain their stress-strain curves and relevant mechanical properties after cooling them down from different elevated temperatures. It was found that the post-fire mechanical properties of cold-formed steels are different to the original ambient temperature mechanical properties. Hence a new set of equations is proposed to predict the reduced mechanical properties of cold-formed steels after a fire event.

Design of cold-formed steel columns at elevated temperatures subject to flexural-torsional buckling

Cold-formed steel members are often subject to axial compression loads in a range of applications. These thin-walled members can be subject to various types of buckling modes, including flexural-torsional buckling. Design standards provide guidelines for columns subject to flexural-torsional buckling modes at ambient temperature. However, there are no specific design guidelines for elevated temperature conditions. Hence extensive research efforts have gone into the many investigations addressing the flexural-torsional buckling behaviour of cold-formed steel columns at elevated temperatures.This research has reviewed the accuracy of the current design rules in AS/NZS 4600 and the North American Specification in determining the member capacities of cold-formed steel columns using the results from detailed finite element analyses and an experimental study of lipped channel columns. It was found that the current ambient temperature Australian and American design rules accurately predicted the member capacities of pin ended lipped channel columns undergoing flexural torsional buckling at elevated temperatures by simply using the appropriate elevated temperature mechanical properties. However, for fixed ended columns with warping fixity undergoing flexural-torsional buckling, it was found that the current design rules significantly underestimated the column capacities as they disregard the beneficial effect of warping fixity. This research has therefore proposed improved design rules and verified their accuracy using finite element analysis and test results of cold-formed lipped channel columns made of three cross-sections and five different steel grades and thicknesses. This paper presents the details of this research study and the results.

Buckling tests of fire exposed cold-formed steel columns

Cold-formed steel sections are commonly used in low-rise commercial and residential buildings. During fire events, cold-formed steel structural elements in these buildings will be exposed to elevated temperatures. Hence after such events there is a need to evaluate the residual strength of these structural elements. However, only limited information is available in relation to the residual strength of fire exposed cold-formed steel sections. This means conservative decisions are often made in relation to fire exposed building structures. This research is aimed at investigating the buckling capacities of fire exposed cold-formed lipped channel steel sections. A series of compression tests of fire exposed, short lipped channel columns made of varying steel grades and thicknesses was undertaken in this research. Test columns were first exposed to different elevated temperatures up to 800 oC. They were then allowed to cool down at ambient temperatures before they were tested to failure. Similarly tensile coupon tests were also undertaken after being exposed to various elevated temperatures, from which the residual mechanical properties (yield stress and Young’s modulus) of the steels used in this study were derived. Using these mechanical properties, the residual compression capacities of tested short columns were predicted using the currently used design rules in AS/NZS 4600 and AISI cold-formed steel standards. This comparison showed that ambient temperature design rules for compression members can be used to predict the residual compression capacities of fire exposed short or laterally restrained cold-formed steel columns provided the maximum temperature experienced by the columns can be estimated after a fire event. Such residual capacity assessments will allow structural and fire engineers to make an accurate prediction of the safety of fire exposed buildings. This paper presents the details of this experimental study and the results.

Distortional buckling behaviour of fire exposed cold-formed steel columns

Cold-formed steel sections are commonly used in low-rise commercial and residential buildings. During fire events, cold-formed steel structural elements in these buildings can be exposed to elevated temperatures. Hence after such events there is a need to evaluate their residual strengths. However, only limited information is available in relation to the residual strength of fire exposed cold-formed steel sections. This research is aimed at investigating the distortional buckling capacities of fire exposed cold-formed lipped channel sections. A series of compression tests of fire exposed, short lipped channel columns made of varying steel grades and thicknesses was undertaken in this research. Test columns were first exposed to different elevated temperatures up to 800 oC, and then tested to failure after cooling down. Suitable finite element models were developed with post-fire mechanical properties to simulate the behaviour of tested columns and were validated using test results. The residual compression capacities of short columns were also predicted using the current cold-formed steel standards and compared with test and finite element analysis results. This comparison showed that ambient temperature design rules for columns can be used to predict the residual compression capacities of fire exposed short or laterally restrained cold-formed steel columns provided the maximum temperature experienced by the column can be estimated after a fire event. Such residual capacity assessments will allow engineers to evaluate the safety of fire exposed buildings. This paper presents the details of this experimental study, finite element analyses and the results.

Molecular analysis of the acinetobacter baumannii biofilm-associated protein

Acinetobacter baumannii is a multidrug-resistant pathogen associated with hospital outbreaks of infection across the globe, particularly in the intensive care unit. The ability of A. baumannii to survive in the hospital environment for long periods is linked to antibiotic resistance and its capacity to form biofilms. Here we studied the prevalence, expression, and function of the A. baumannii biofilm-associated protein (Bap) in 24 carbapenem-resistant A. baumannii ST92 strains isolated from a single institution over a 10-year period. The bap gene was highly prevalent, with 22/24 strains being positive for bap by PCR. Partial sequencing of bap was performed on the index case strain MS1968 and revealed it to be a large and highly repetitive gene approximately 16 kb in size. Phylogenetic analysis employing a 1,948-amino-acid region corresponding to the C terminus of Bap showed that BapMS1968 clusters with Bap sequences from clonal complex 2 (CC2) strains ACICU, TCDC-AB0715, and 1656-2 and is distinct from Bap in CC1 strains. By using overlapping PCR, the bapMS1968 gene was cloned, and its expression in a recombinant Escherichia coli strain resulted in increased biofilm formation. A Bap-specific antibody was generated, and Western blot analysis showed that the majority of A. baumannii strains expressed an ∼200-kDa Bap protein. Further analysis of three Bap-positive A. baumannii strains demonstrated that Bap is expressed at the cell surface and is associated with biofilm formation. Finally, biofilm formation by these Bap-positive strains could be inhibited by affinity-purified Bap antibodies, demonstrating the direct contribution of Bap to biofilm growth by A. baumannii clinical isolates.

Functional heterogeneity of the UpaH autotransporter protein from Uropathogenic Escherichia coli

Uropathogenic Escherichia coli (UPEC) is responsible for the majority of urinary tract infections (UTI). To cause a UTI, UPEC must adhere to the epithelial cells of the urinary tract and overcome the shear flow forces of urine. This function is mediated primarily by fimbrial adhesins, which mediate specific attachment to host cell receptors. Another group of adhesins that contributes to UPEC-mediated UTI is autotransporter (AT) proteins. AT proteins possess a range of virulence properties, such as adherence, aggregation, invasion, and biofilm formation. One recently characterized AT protein of UPEC is UpaH, a large adhesin-involved-in-diffuse-adherence (AIDA-I)-type AT protein that contributes to biofilm formation and bladder colonization. In this study we characterized a series of naturally occurring variants of UpaH. We demonstrate that extensive sequence variation exists within the passenger-encoding domain of UpaH variants from different UPEC strains. This sequence variation is associated with functional heterogeneity with respect to the ability of UpaH to mediate biofilm formation. In contrast, all of the UpaH variants examined retained a conserved ability to mediate binding to extracellular matrix (ECM) proteins. Bioinformatic analysis of the UpaH passenger domain identified a conserved region (UpaHCR) and a hydrophobic region (UpaHHR). Deletion of these domains reduced biofilm formation but not the binding to ECM proteins. Despite variation in the upaH sequence, the transcription of upaH was repressed by a conserved mechanism involving the global regulator H-NS, and mutation of the hns gene relieved this repression. Overall, our findings shed new light on the regulation and functions of the UpaH AT protein.

Discovery of an archetypal protein transport system in bacterial outer membranes

Bacteria have mechanisms to export proteins for diverse purposes, including colonization of hosts and pathogenesis. A small number of archetypal bacterial secretion machines have been found in several groups of bacteria and mediate a fundamentally distinct secretion process. Perhaps erroneously, proteins called 'autotransporters' have long been thought to be one of these protein secretion systems. Mounting evidence suggests that autotransporters might be substrates to be secreted, not an autonomous transporter system. We have discovered a new translocation and assembly module (TAM) that promotes efficient secretion of autotransporters in proteobacteria. Functional analysis of the TAM in Citrobacter rodentium, Salmonella enterica and Escherichia coli showed that it consists of an Omp85-family protein, TamA, in the outer membrane and TamB in the inner membrane of diverse bacterial species. The discovery of the TAM provides a new target for the development of therapies to inhibit colonization by bacterial pathogens.

Characterisation of a cell wall-anchored protein of Staphylococcus saprophyticus associated with linoleic acid resistance

The Gram-positive bacterium Staphylococcus saprophyticus is the second most frequent causative agent of community-acquired urinary tract infections (UTI), accounting for up to 20% of cases. A common feature of staphylococci is colonisation of the human skin. This involves survival against innate immune defenses including antibacterial unsaturated free fatty acids such as linoleic acid which act by disrupting bacterial cell membranes. Indeed, S. saprophyticus UTI is usually preceded by perineal skin colonisation.

Characterization of EhaJ, a new autotransporter protein from enterohemorrhagic and enteropathogenic Escherichia coli

Enterohemorrhagic Escherichia coli (EHEC) and enteropathogenic E. coli (EPEC) are diarrheagenic pathotypes of E. coli that cause gastrointestinal disease with the potential for life-threatening sequelae. While certain EHEC and EPEC virulence mechanisms have been extensively studied, the factors that mediate host colonization remain to be properly defined. Previously, we identified four genes (ehaA, ehaB, ehaC, and ehaD) from the prototypic EHEC strain EDL933 that encode for proteins that belong to the autotransporter (AT) family. Here we have examined the prevalence of these genes, as well as several other AT-encoding genes, in a collection of EHEC and EPEC strains. We show that the complement of AT-encoding genes in EHEC and EPEC strains is variable, with some AT-encoding genes being highly prevalent. One previously uncharacterized AT-encoding gene, which we have termed ehaJ, was identified in 12/44 (27%) of EHEC and 2/20 (10%) of EPEC strains. The ehaJ gene lies immediately adjacent to a gene encoding a putative glycosyltransferase (referred to as egtA). Western blot analysis using an EhaJ-specific antibody indicated that EhaJ is glycosylated by EgtA. Expression of EhaJ in a recombinant E. coli strain, revealed EhaJ is located at the cell surface and in the presence of the egtA glycosyltransferase gene mediates strong biofilm formation in microtiter plate and flow cell assays. EhaJ also mediated adherence to a range of extracellular matrix proteins, however this occurred independent of glycosylation. We also demonstrate that EhaJ is expressed in a wild-type EPEC strain following in vitro growth. However, deletion of ehaJ did not significantly alter its adherence or biofilm properties. In summary, EhaJ is a new glycosylated AT protein from EPEC and EHEC. Further studies are required to elucidate the function of EhaJ in colonization and virulence.

UpaH Is a newly identified autotransporter protein That contributes to biofilm formation and bladder colonization by Uropathogenic Escherichia coli CFT073

Escherichia coli is the primary cause of urinary tract infection (UTI) in the developed world. The major factors associated with virulence of uropathogenic E. coli (UPEC) are fimbrial adhesins, which mediate specific attachment to host receptors and trigger innate host responses. Another group of adhesins is represented by the autotransporter (AT) subgroup of proteins. In this study, we identified a new AT-encoding gene, termed upaH, present in a 6.5-kb unannotated intergenic region in the genome of the prototypic UPEC strain CFT073. Cloning and sequencing of the upaH gene from CFT073 revealed an intact 8.535-kb coding region, contrary to the published genome sequence. The upaH gene was widely distributed among a large collection of UPEC isolates as well as the E. coli Reference (ECOR) strain collection. Bioinformatic analyses suggest β-helix as the predominant structure in the large N-terminal passenger (α) domain and a 12-strand β-barrel for the C-terminal β-domain of UpaH. We demonstrated that UpaH is expressed at the cell surface of CFT073 and promotes biofilm formation. In the mouse UTI model, deletion of the upaH gene in CFT073 and in two other UPEC strains did not significantly affect colonization of the bladder in single-challenge experiments. However, in competitive colonization experiments, CFT073 significantly outcompeted its upaH isogenic mutant strain in urine and the bladder.

The Escherichia coli O157:H7 EhaB autotransporter protein binds to laminin and collagen I and induces a serum IgA response in O157:H7 challenged cattle

Enterohaemorrhagic Escherichia coli (EHEC) are a subgroup of Shiga toxin-producing E. coli that cause gastrointestinal disease with the potential for life-threatening sequelae. Cattle serve as the natural reservoir for EHEC and outbreaks occur sporadically as a result of contaminated beef and other farming products. While certain EHEC virulence mechanisms have been extensively studied, the factors that mediate host colonization are poorly defined. Previously, we identified four proteins (EhaA,B,C,D) from the prototypic EHEC strain EDL933 that belong to the autotransporter (AT) family. Here we characterize the EhaB AT protein. EhaB was shown to be located at the cell surface and overexpression in E. coli K-12 resulted in significant biofilm formation under continuous flow conditions. Overexpression of EhaB in E. coli K12 and EDL933 backgrounds also promoted adhesion to the extracellular matrix proteins collagen I and laminin. An EhaB-specific antibody revealed that EhaB is expressed in E. coli EDL933 following in vitro growth. EhaB also cross-reacted with serum IgA from cattle challenged with E. coli O157:H7, indicating that EhaB is expressed in vivo and elicits a host IgA immune response.