A quantitative assessment of the extent and distribution of textile fibre transfer to persons involved in physical assault.

Knowledge of the number of fibres transferred during a particular activity is essential for the interpretation of findings in similar criminal cases. In this regard, violent contacts and physical assaults still present a challenge, due to a lack of robust published data. Hereby, we present the outcome of an empirical study where different assault activities were simulated by a Jiu Jitsu team and participants were asked to play either the role of an aggressive 'assailant' or a defensive 'victim', wearing cotton garments (i.e., Gi's). Four different scenarios were simulated in replicates (n = 5), each of them involving different intensity levels (low and high) and duration times (30 and 60 s). Results showed that approximately 1,000 to 44,000 fibres were cross-transferred between the participants' garments, with noticeable differences between the different scenarios. These values were significantly larger than those published in previous studies and, therefore, suggested the possibility of a current underestimation of the number of fibres transferred in physical assaults. Furthermore, statistical analysis by ANOVA indicated that the all the variables tested (i.e., intensity level, duration time, and participants role) had a significant effect on the number of transferred fibres (p < 0.001) and, consequently, that some knowledge of the case circumstances may be important to make more educated estimations. This is the first time that such a methodology has been applied for the quantitative assessment of fibre transfer between participants in assault activities. Data are expected to help practitioners with the interpretation of findings in real casework and lead to a more robust evidential assessment.

Simple detection of protein soft structure changes.

We present a rapid method for protein tertiary structure analysis which avoids the need for techniques such as circular dichroism and differential scanning calorimetry. Small changes to a protein's noncovalent "soft" structure are detected by exploiting differences in thermal stability and fluorescent reporter binding. It can detect subtle stability differences using micrograms of protein in 2 microL volumes within minutes.

Self-recognition by an intrinsically disordered protein.

The intrinsically disordered translocation domain (T-domain) of the protein antibiotic colicin N binds to periplasmic receptors of target Escherichia coli cells in order to penetrate their inner membranes. We report here that the specific 27 consecutive residues of the T-domain of colicin N known to bind to the helper protein TolA in target cells also interacts intramolecularly with folded regions of colicin N. We suggest that this specific self-recognition helps intrinsically disordered domains to bury their hydrophobic recognition motifs and protect them against degradation, showing that an impaired self-recognition leads to increased protease susceptibility.

Unfolding transitions of Bacillus anthracis protective antigen.

Protective antigen (PA) is an 83kDa protein which, although essential for toxicity of Bacillus anthracis, is harmless and an effective vaccine component. In vivo it undergoes receptor binding, proteolysis, heptamerisation and membrane insertion. Here we probe the response of PA to denaturants, temperature and pH. We present analyses (including barycentric mean) of the unfolding and refolding behavior of PA and reveal the origin of two critical steps in the denaturant unfolding pathway in which the first step is a calcium and pH dependent rearrangement of domain 1. Thermal unfolding fits a single transition near 50 degrees C. We show for the first time circular dichroism (CD) spectra of the heptameric, furin-cleaved PA63 and the low-pH forms of both PA83 and PA63. Although only PA63 should reach the acidic endosome, both PA83 and PA63 undergo similar acidic transitions and an unusual change from a beta II to a beta I CD spectrum.

Determining OMP topology by computation, surface plasmon resonance and cysteine labelling: the test case of OMPG.

Bacterial outer-membrane proteins (OMP) are important in pathogenicity and the recently solved structure of OmpG provides an excellent test case for topological predictions since it is monomeric. Here we compare the results of applying several computerised structure prediction algorithms to the sequence of OmpG. Furthermore, we probe the OmpG topology by both an established chemical labelling approach and a new method which combines epitope insertion and surface plasmon resonance. The computational approaches are broadly accurate but the exact choice of the number of beta strands remains difficult. The algorithms also tend to predict the entire beta strand rather than just the transmembrane region. Epitope insertion clearly pinpoints exposed loops but its utility in defining buried or periplasmic sites is less clear cut. Cysteine-mutant labelling is largely confined to exposed residues but one periplasmic cysteine may be labelled by reagents entering via the OmpG pore.

Immunogenicity of a Yersinia pestis vaccine antigen monomerized by circular permutation.

Caf1, a chaperone-usher protein from Yersinia pestis, is a major protective antigen in the development of subunit vaccines against plague. However, recombinant Caf1 forms polymers of indeterminate size. We report the conversion of Caf1 from a polymer to a monomer by circular permutation of the gene. Biophysical evaluation confirmed that the engineered Caf1 was a folded monomer. We compared the immunogenicity of the engineered monomer with polymeric Caf1 in antigen presentation assays to CD4 T-cell hybridomas in vitro, as well as in the induction of antibody responses and protection against subcutaneous challenge with Y. pestis in vivo. In C57BL/6 mice, for which the major H-2(b)-restricted immunodominant CD4 T-cell epitopes were intact in the engineered monomer, immunogenicity and protective efficacy were preserved, although antibody titers were decreased 10-fold. Disruption of an H-2(d)-restricted immunodominant CD4 T-cell epitope during circular permutation resulted in a compromised T-cell response, a low postvaccination antibody titer, and a lack of protection of BALB/c mice. The use of circular permutation in vaccine design has not been reported previously.

Refolding of Escherichia coli outer membrane protein F in detergent creates LPS-free trimers and asymmetric dimers.

The Escherichia coli OmpF (outer-membrane protein F; matrix porin) is a homotrimeric beta-barrel and a member of the bacterial porin superfamily. It is the best characterized porin protein, but has resisted attempts to refold it efficiently in vitro. In the present paper, we report the discovery of detergent-based folding conditions, including dodecylglucoside, which can create pure samples of trimeric OmpF. Whereas outer membrane LPS (lipopolysaccharide) is clearly required for in vivo folding, the artificially refolded and LPS-free trimer has properties identical with those of the outer-membrane-derived form. Thus LPS is not required either for in vitro folding or for structural integrity. Dimeric forms of OmpF have been observed in vivo and are proposed to be folding intermediates. In vitro, dimers occur transiently in refolding of trimeric OmpF and, in the presence of dodecylmaltoside, pure dimer can be prepared. This form has less beta-structure by CD and shows lower thermal stability than the trimer. Study of these proteins at the single-molecule level is possible because each OmpF subunit forms a distinct ion channel. Whereas each trimer contains three channels of equal conductance, each dimer always contains two distinct channel sizes. This provides clear evidence that the two otherwise identical monomers adopt different structures in the dimer and indicates that the asymmetric interaction, characteristic of C3 symmetry, is formed at the dimer stage. This asymmetric dimer may be generally relevant to the folding of oligomeric proteins with odd numbers of subunits such as aspartate transcarbamoylase.