Injury Risk Functions for the Midsized Male Wrist and Elbow as a Result of Behind Shield Blunt Trauma.

Ballistic shields protect users from a variety of threats, including projectiles. Shield back-face deformation (BFD) is the result of the shield deflecting or absorbing a projectile and deforming toward the user. Back-face deformation can result in localized blunt loading to the upper extremity, where the shield is supported by the user. Two vulnerable locations along the upper extremity were investigated-the wrist and elbow-on eight postmortem human subjects (PMHS) using a pneumatic impacting apparatus for investigating the fracture threshold as a result of behind shield blunt trauma (BSBT). Impacting parameters were established by subjecting an augmented WorldSID anthropomorphic test device (ATD) positioned behind a ballistic shield to ballistic impacts. These data were used to form the impact parameters applied to PMHS, where the wrist most frequently fractured at the distal radius and the elbow most frequently fractured at the radial head. The fracture threshold for the wrist was 5663±1386 N (mean±standard deviation), higher than the elbow at 4765±894 N (though not significantly, p = 0.15). The failure impact velocity for wrist impacts was 17.7±2.1 m/s, while for the elbow, the failure impact velocity was 19.5±0.9 m/s. An approximate 10% risk of fracture threshold was identified on the modified WorldSID ATD (no flesh analogue included) to inform future protective standards.

Injury Risk for the Hand and Forearm Under Loading Representative of Behind Shield Blunt Trauma.

Ballistic shields protect users from a variety of threats, including projectiles. Shield back-face deformation (BFD) is the result of the shield absorbing energy from a projectile and deforming towards the user. Back-face deformation can result in localized blunt loading to the upper extremity, where the shield is supported by the user and may cause injury through behind armour blunt trauma (BABT) mechanisms. Post-mortem human subject (PMHS) responses are critical to identify the injury risk in these high-rate scenarios and are used to quantify the injury tolerance. Two vulnerable locations along the upper extremity were investigated-the hand and forearm-using eight PMHS to identify the fracture threshold resulting from shield BABT loading conditions. Impacts delivered to the hand at 16.4 ± 0.8 m/s resulted in failure loads of 3818 ± 897 N, whilst the forearm impacts delivered at a similar velocity of 16.9 ± 1.9 m/s had lower failure loads at 3011 ± 656 N. The corresponding 10% risk of hand and forearm fractures (as measured on a modified WorldSID Anthropomorphic Test Device) were identified as 11.0 kN and 8.1 kN, respectively, which should be used when evaluating future designs of composite ballistic shields. This study is the first known investigation of the upper extremity to this high loading rate scenario and provides the foundation for future biomechanical research in the area of behind shield blunt trauma.

Mycoplasma bovis testing for the screening of semen imported into New Zealand.

AIMS: To evaluate the fitness of three PCR assays for the detection of Mycoplasma bovis in dilute (extended) bovine semen, and a reverse transcriptase-PCR (RT-PCR) adaptation as a proxy for viability. MATERIALS AND METHODS: Four commercial kit-based methods for nucleic acid extraction were compared to test for the presence of PCR inhibitors in nucleic acid extracted from undiluted and diluted semen. Then, analytical sensitivity, analytical specificity, and diagnostic specificity of two real-time PCR and one conventional PCR were evaluated for the detection of M. bovis DNA in semen and compared against microbial culture. Furthermore, an RT-PCR was adapted to detect RNA only and tested on viable and non-viable M. bovis to establish its ability to discriminate between the two. RESULTS: No significant PCR inhibition was detected from the dilute semen. All DNA extraction methods except one were equivalent, regardless of semen dilution. The analytical sensitivity of the real-time PCR assays was estimated as 45.6 cfu per 200 µL semen straw (2.2 × 102 cfu/mL). The conventional PCR was 10 times less sensitive. No cross-reactivity was observed for the real-time PCR for any of the bacteria tested and the diagnostic specificity was estimated as 100 (95% CI = 94.04-100) %. The RT-PCR was poor in distinguishing between viable and non-viable M. bovis. The mean quantification cycle (Cq) values for RNA extracted from different treatments to kill M. bovis remained unchanged 0-48 hours after inactivation. CONCLUSION AND CLINICAL RELEVANCE: The real-time PCR were fit for the purpose of screening dilute semen for the detection of M. bovis to prevent incursion via importation of infected semen. The real-time PCR assays can be used interchangeably. The RT-PCR could not reliably indicate the viability of M. bovis. Based on the results from this study, a protocol and guidelines have been produced for laboratories elsewhere that wish to test bovine semen for M. bovis.

Behind Shield Blunt Trauma: Characterizing the Back-Face Deformation of Shields with a Focus on Upper Limb Loading.

Shield back-face deformation (BFD) is the result of composite ballistic shields deflecting or absorbing a projectile's energy and deforming towards the user. BFD can result in localized loading to the upper extremity, where the shield is secured to the user. An augmented anthropomorphic test device upper extremity was used to quantify this applied load. Four locations along the upper extremity were tested-the hand, wrist, forearm, and elbow-for investigating differing boundary conditions and their effect on resultant load. Varying stand-off distances, the distance between the back of the shield and the force sensor, were investigated. Digital image correlation was also conducted to measure the dynamic displacement of the shield. The mean peak back-face velocity of the shield was 208.4 ± 38.8 m/s, while the average affected area was 1505 ± 158.3 mm2. Impulse was not significantly affected by anatomical location for the same stand-off distance; however, as stand-off distance decreased, the measured force significantly increased (p < 0.05). Notably, impact duration did not differ significantly for any of the impact scenarios. This is the first step in developing injury criteria for this region resulting from behind shield blunt trauma, and these data will be used for developing injury thresholds in post-mortem human surrogates.

Ovine FABP4 Variation and Its Association With Flystrike Susceptibility.

Flystrike is a major cost and a welfare issue for the New Zealand sheep industry. There are several factors that can predispose sheep to flystrike, such as having fleecerot, a urine-stained breech, and "dags" (an accumulation of fecal matter in the wool of the breech). The FABP4 gene (FABP4) has been associated with variation in ovine fleecerot resistance, with a strong genetic correlation existing between fleecerot and flystrike occurrence. In this study, blood samples were collected from sheep with and without flystrike for DNA typing. PCR-SSCP analyses were used to genotype two regions of ovine FABP4. Sheep with the A 1 variant of FABP4 were found to be less likely (odds ratio 0.689, P = 0.014) to have flystrike than those without A 1. The likelihood of flystrike occurrence decreased as copy number of A 1 increased (odds ratio 0.695, P = 0.006). This suggests that FABP4 might be a candidate gene for flystrike resilience in sheep, although further research is required to verify this association.

Conserved, unstructured regions in Pseudomonas aeruginosa PilO are important for type IVa pilus function.

Pseudomonas aeruginosa uses long, thin fibres called type IV pili (T4P) for adherence to surfaces, biofilm formation, and twitching motility. A conserved subcomplex of PilMNOP is required for extension and retraction of T4P. To better understand its function, we attempted to co-crystallize the soluble periplasmic portions of PilNOP, using reductive surface methylation to promote crystal formation. Only PilOΔ109 crystallized; its structure was determined to 1.7 Å resolution using molecular replacement. This new structure revealed two novel features: a shorter N-terminal α1-helix followed by a longer unstructured loop, and a discontinuous ß-strand in the second αßß motif, mirroring that in the first motif. PISA analysis identified a potential dimer interface with striking similarity to that of the PilO homolog EpsM from the Vibrio cholerae type II secretion system. We identified highly conserved residues within predicted unstructured regions in PilO proteins from various Pseudomonads and performed site-directed mutagenesis to assess their role in T4P function. R169D and I170A substitutions decreased surface piliation and twitching motility without disrupting PilO homodimer formation. These residues could form important protein-protein interactions with PilN or PilP. This work furthers our understanding of residues critical for T4aP function.

Independent prescriber physiotherapist led balance clinic: the Southport and Ormskirk pathway.

OBJECTIVE: To report the introduction and impact of non-medical prescribing, initiated to improve patient pathways for those presenting with dizziness and balance disorders. METHODS: The Southport and Ormskirk physiotherapy-led vestibular clinic sees and treats all patients with dizziness and balance disorders referred to the ENT department. Letters are triaged by an audiologist, who also performs an otological examination and hearing test; this is followed by an assessment with the independent prescriber physiotherapist. An ENT consultant is nearby if joint consultation is needed. Diagnoses, treatments and patient satisfaction were studied, with an analysis of the impact of medication management (stopping or starting medicines) on patients and service. RESULTS: In 12 months, 413 new patients with dizziness and balance disorders had appointments. The most common diagnoses were benign paroxysmal positional vertigo and vestibular migraine. Eighty-four per cent of patients required self-management strategies, 50 per cent exercise therapy, 48 per cent medication management and 24 per cent a particle repositioning manoeuvre. Patient satisfaction was high (99 per cent). CONCLUSION: Having an independent prescriber physiotherapist leading the balance clinic has reduced the number of hospital visits and onward referrals. Nearly half of all patients required medication management as part of their dizziness or balance treatment.

Association between variation in faecal egg count for a natural mixed field-challenge of nematode parasites and TLR4 variation.

Research has shown that Toll-like receptor 4 (TLR4) is important in immune responses to some helminth parasites. In sheep, variation in the PAMP region of TLR4 may result in structurally and thus functionally different TLR4 molecules, and this may consequently lead to variation in the TLR4 response to parasite infections. This study involved three separate, but related sheep breeds (Merino, Polwarth and Corriedale sheep) and a total of 885 lambs from five New Zealand farms that underwent a mixed field-challenge from gastro-intestinal parasites. Faecal samples were collected at approximately 4 and 9 months of age and faecal egg counts (FECs) for Nematodirus spp. and Strongyle species determined, along with the total number of eggs per gram (EPG). Analysis of the five farms collectively revealed an association (P=0.023) between the presence of TLR4 variant *02 (mean 24 EPG) and the absence of the variant (mean 32 EPG) at 9 months of age. Conversely the presence of *03 had a significantly (P=0.047) higher mean Nematodirus spp. FEC (mean 42 EPG) compared to the absence (mean 28 EPG) at 9 months of age. More associations were revealed when the data were split according to the dominant faecal parasite species. With a predominantly Trichostrongylus spp. FEC group of lambs at 9 months of age, the presence of TLR4 variant *02 was found to have significantly (P=0.003) lower Nematodirus spp. FEC (mean 4 EPG), and also significantly (P=0.033) lower total FEC (mean 312 EPG) when compared to sheep without the variant (mean 15 EPG and 449 EPG, respectively). The presence of TLR4 variant *03 and *04 were associated or tended to be associated (P=0.010 and P=0.088, respectively) with higher Nematodirus spp. FEC (mean 25 EPG and 22 EPG, respectively) when compared to lambs without the variant (mean 10 EPG and 11 EPG, respectively). These results suggest that TLR4 variation may be affecting the immune response to gastro-intestinal parasites in sheep, although principally to Nematodirus spp. infections and not Strongyle species infections.

Recurrent life-threatening reactions to platelet transfusion in an aplastic anaemia patient with a paroxysmal nocturnal haemoglobinuria clone.

A 60-year-old woman was diagnosed with non-severe aplastic anaemia when she presented with anaemia and thrombocytopenia. She developed recurrent life-threatening hypotensive reactions during transfusion of leukodepleted platelet concentrates, and washed platelet concentrates prevented the development of such reactions subsequently. A paroxysmal nocturnal haemoglobinuria clone was detected on investigating for aplastic anaemia, which has been speculated to play a role in the recurrent hypotensive reactions.

An atypical genotype of Toxoplasma gondii as a cause of mortality in Hector's dolphins (Cephalorhynchus hectori).

Hector's dolphins (Cephalorhynchus hectori) are a small endangered coastal species that are endemic to New Zealand. Anthropogenic factors, particularly accidental capture in fishing nets, are believed to be the biggest threat to survival of this species. The role of infectious disease as a cause of mortality has not previously been well investigated. This study investigates Toxoplasma gondii infection in Hector's dolphins, finding that 7 of 28 (25%) dolphins examined died due to disseminated toxoplasmosis, including 2 of 3 Maui's dolphins, a critically endangered sub-species. A further 10 dolphins had one or more tissues that were positive for the presence of T. gondii DNA using PCR. Genotyping revealed that 7 of 8 successfully amplified isolates were an atypical Type II genotype. Fatal cases had necrotising and haemorrhagic lesions in the lung (n=7), lymph nodes (n=6), liver (n=4) and adrenals (n=3). Tachyzoites and tissue cysts were present in other organs including the brain (n=5), heart (n=1), stomach (n=1) and uterus (n=1) with minimal associated inflammatory response. One dolphin had a marked suppurative metritis in the presence of numerous intra-epithelial tachyzoites. No dolphins had underlying morbillivirus infection. This study provides the first evidence that infectious agents could be important in the population decline of this species, and highlights the need for further research into the route of entry of T. gondii organisms into the marine environment worldwide.

Characterization of the PilN, PilO and PilP type IVa pilus subcomplex.

Type IVa pili are bacterial nanomachines required for colonization of surfaces, but little is known about the organization of proteins in this system. The Pseudomonas aeruginosa pilMNOPQ operon encodes five key members of the transenvelope complex facilitating pilus function. While PilQ forms the outer membrane secretin pore, the functions of the inner membrane-associated proteins PilM/N/O/P are less well defined. Structural characterization of a stable C-terminal fragment of PilP (PilP(Δ71)) by NMR revealed a modified ß-sandwich fold, similar to that of Neisseria meningitidis PilP, although complementation experiments showed that the two proteins are not interchangeable likely due to divergent surface properties. PilP is an inner membrane putative lipoprotein, but mutagenesis of the putative lipobox had no effect on the localization and function of PilP. A larger fragment, PilP(Δ18-6His), co-purified with a PilN(Δ44)/PilO(Δ51) heterodimer as a stable complex that eluted from a size exclusion chromatography column as a single peak with a molecular weight equivalent to two heterotrimers with 1:1:1 stoichiometry. Although PilO forms both homodimers and PilN-PilO heterodimers, PilP(Δ18-6His) did not interact stably with PilO(Δ51) alone. Together these data demonstrate that PilN/PilO/PilP interact directly to form a stable heterotrimeric complex, explaining the dispensability of PilP's lipid anchor for localization and function.

Utilization of dried blood spots within drug discovery: modification of a standard DiLab(®) AccuSampler(®) to facilitate automatic dried blood spot sampling.

The use of dried blood spots (DBS) in preclinical studies has seen an enormous increase over the past two years. Despite its positive impact on the 3Rs (reduce, replace and refine), its uptake in exploratory drug discovery has been limited due mainly to protracted method development time in bioanalysis but also the need for small volumes (<20 µL) to be sampled manually. Automatic blood sampling technology such as the DiLab(®) AccuSampler(®) is widely used in drug discovery to facilitate exploratory rodent-based pharmacokinetic and pharmacokinetic/pharmacodynamic studies with minimal animal handling. Propranolol was orally administered to a Han-Wistar rat attached to either a standard DiLab(®) AccuSampler(®) or a retrofitted unit designed to directly collect the DBS samples. In all, 50 or 20 µL blood samples were then collected via the standard or retrofitted unit, respectively, at six timepoints over a 7 h period. After drying and storage the DBS samples were analysed for propranolol via liquid chromatography-mass spectrometry. In this report we demonstrate that a standard DiLab(®) AccuSampler(®) can be easily retrofitted to facilitate automatic dried blood spot sampling and that time-concentration data generated from these samples are equivalent to that from manually spotted samples.

Revised UK guidelines for the management of cutaneous melanoma 2010.

These guidelines for the management of cutaneous melanoma present an evidence-based guidance for treatment, with identification of the strength of evidence available at the time of preparation of the guidelines, and a brief overview of epidemiology, diagnosis, investigation, and follow-up.

Situational analysis of varying models of adherence support and loss to follow up rates; findings from 27 treatment facilities in eight resource limited countries.

OBJECTIVES: Large-scale provision of ART in the absence of viral load monitoring, resistance testing, and limited second-line treatment options places adherence support as a vital therapeutic intervention. We aimed to compare patient loss to follow up rates with the degree of adherence support through a retrospective review of patients enrolled in the AIDSRelief program between August 2004 and June 2005. METHODS: Loss to follow up data were analysed and programs were categorised into one of four tiered levels of adherence support models: Tier I, II, III, and IV which increase from lowest to highest support. Bivariate and t-test analyses were used to test for significant differences between the models. RESULTS: 13,391 patients at 27 treatment facilities from six African and two Caribbean countries began antiretroviral therapy within the first year of the AIDSRelief program. The mean loss to follow up within the first year was 7.5%. Eight facilities were Tier I, three (Tier II), nine (Tier III), and seven (Tier IV). Facilities in Tier I had a loss to follow up rate of 14%, Tier II (10%), Tier III (5%), and Tier IV (1%). The proportion of loss to follow up for Tier I and Tier III were significantly different from each other (P < 0.02), as were Tier I and Tier IV (P < 0.006). There were differences between Tier II and Tier IV (P < 0.009) as well as Tier III and Tier IV (P < 0.017). CONCLUSION: These data strongly support the use of proactive adherence support programs, beyond routine patient counselling and defaulter tracking to support the'public health approach'to ART.

A survey of the management of needlestick injuries from incapacitated patients in intensive care units.

The Human Tissue Act 2004 and Mental Capacity Act 2005 resulted in a change in the management of needlestick injuries sustained from incapacitated patients. It appears unlawful to test for blood-borne viruses without a patient's consent for the sole benefit of the healthcare worker. This survey of intensive care units within England, Wales and Northern Ireland investigated how needlestick injuries from incapacitated patients had been managed within the previous year. Of the 225 intensive care units surveyed, 99 (44%) responded. Sixty-two (62.6%) reported a needlestick injury to a healthcare worker from an incapacitated patient. Thirty-six (64.3%) patients were tested for blood-borne viruses without consent. Sixteen (25.8%) patients tested positive for blood-borne viruses. Only 19 (30.6%) healthcare workers took post-exposure prophylaxis following the injury. These results show that needlestick injuries from incapacitated patients are common and that the majority of patients were tested for blood-borne viruses without consent.

PilM/N/O/P proteins form an inner membrane complex that affects the stability of the Pseudomonas aeruginosa type IV pilus secretin.

The highly conserved pilM/N/O/P/Q gene cluster is among the core set of genes required for cell surface expression of type IV pili and associated twitching motility. With the exception of the outer membrane secretin, a multimer of PilQ subunits, the specific functions of the products encoded by this gene cluster are poorly characterized. Orthologous proteins in the related bacterial type II secretion system have been shown to interact to form an inner membrane complex required for protein secretion. In this study, we provide evidence that the PilM/N/O/P proteins form a functionally equivalent type IVa pilus complex. Using Pseudomonas aeruginosa as model organism, we found that all four proteins, including the nominally cytoplasmic PilM, colocalized to the inner membrane. Stability studies via Western blot analyses revealed that loss of one component has a negative impact on the levels of other members of the putative complex. Furthermore, complementation studies revealed that the stoichiometry of the components is important for the correct formation of a stable complex in vivo. We provide evidence that an intact inner membrane complex is required for optimal formation of the outer membrane complex of the type IVa pilus system in P. aeruginosa, as PilQ stability is negatively affected in its absence. Finally, we show that, in the absence of the pilin subunit, the levels of membrane-bound components of the inner membrane complex are negatively regulated by the PilR/S two-component system, suggesting a role for PilR/S in sensing the piliation status of the cell.

Periplasmic domains of Pseudomonas aeruginosa PilN and PilO form a stable heterodimeric complex.

Type IV pili (T4P) are bacterial virulence factors responsible for attachment to surfaces and for twitching motility, a motion that involves a succession of pilus extension and retraction cycles. In the opportunistic pathogen Pseudomonas aeruginosa, the PilM/N/O/P proteins are essential for T4P biogenesis, and genetic and biochemical analyses strongly suggest that they form an inner-membrane complex. Here, we show through co-expression and biochemical analysis that the periplasmic domains of PilN and PilO interact to form a heterodimer. The structure of residues 69-201 of the periplasmic domain of PilO was determined to 2.2 A resolution and reveals the presence of a homodimer in the asymmetric unit. Each monomer consists of two N-terminal coiled coils and a C-terminal ferredoxin-like domain. This structure was used to generate homology models of PilN and the PilN/O heterodimer. Our structural analysis suggests that in vivo PilN/O heterodimerization would require changes in the orientation of the first N-terminal coiled coil, which leads to two alternative models for the role of the transmembrane domains in the PilN/O interaction. Analysis of PilN/O orthologues in the type II secretion system EpsL/M revealed significant similarities in their secondary structures and the tertiary structures of PilO and EpsM, although the way these proteins interact to form inner-membrane complexes appears to be different in T4P and type II secretion. Our analysis suggests that PilN interacts directly, via its N-terminal tail, with the cytoplasmic protein PilM. This work shows a direct interaction between the periplasmic domains of PilN and PilO, with PilO playing a key role in the proper folding of PilN. Our results suggest that PilN/O heterodimers form the foundation of the inner-membrane PilM/N/O/P complex, which is critical for the assembly of a functional T4P complex.

Alginate as an auxiliary bacterial membrane: binding of membrane-active peptides by polysaccharides.

The chronicity of Pseudomonas aeruginosa infections in cystic fibrosis (CF) patients is characterized by overproduction of the exopolysaccharide alginate, in which biofilm bacteria are embedded. Alginate apparently contributes to the antibiotic resistance of bacteria in this form by acting as a diffusion barrier to positively charged antimicrobial agents. We have been investigating cationic antimicrobial peptides (CAPs) (prototypic sequence: KKAAAXAAAAAXAAWAAXAAAKKKK-NH(2), where X is any of the 20 commonly occurring amino acids) that were originally designed as transmembrane mimetic peptides. Peptides of this group above a specific hydrophobicity threshold insert spontaneously into membranes and have antibacterial activity at micromolar concentrations. While investigating the molecular basis of biofilm resistance to peptides, we found that the anionic alginate polysaccharide induces conformational changes in the most hydrophobic of these peptides typically associated with insertion of such peptides into membrane environments [Chan et al., J. Biol. Chem. (2004) vol. 279, pp. 38749-38754]. Through a combination of experiments measuring release of the fluorescent dye calcein from phospholipid vesicles, peptide interactions with vesicles in the presence and absence of alginate, and affinity of peptides for alginate as a function of net peptide core hydrophobicity, we show here that alginate offers a microenvironment that provides a protective mechanism for the encased bacteria by both binding and promoting the self-association of the CAPs. The overall results indicate that hydrophilic alginate polymers contain a significant hydrophobic compartment, and behave as an 'auxiliary membrane' for bacteria, thus identifying a unique protective role for biofilm exopolysaccharide matrices.