In vitro effects of detergent sclerosants on coagulation, platelets and microparticles.

OBJECTIVES: To investigate the in vitro effects of Sodium Tetradecyl Sulphate (STS) and Polidocanol (POL) on clotting tests, clotting factors, platelets and microparticles. MATERIALS AND METHODS: Platelet rich (PRP) and platelet poor (PPP) plasmas were incubated with varying concentrations of STS and POL. Clotting tests, platelet/plasma turbidity, and microparticle studies were performed. Specimens were mixed with individual factor deficient plasmas and clotting factor activities were studied. RESULTS: STS at high concentrations (>0.3%) destroyed platelets, microparticles and the clotting factors V, VII and X. It prolonged all clotting tests including prothrombin time (PT), activated partial thromboplastin time (APTT), non-activated partial thromboplastin time (NAPTT), thrombin time (TT), factor Xa clotting time (XACT) and surface activated clotting time (SACT). Higher concentrations of POL were required to achieve some anticoagulant activity. Low sclerosant concentrations shortened XACT and SACT, and induced release of procoagulant platelet derived microparticles. Increased exposure time resulted in increased procoagulant activity. STS at concentrations higher than 0.5% precipitated a complex containing apolipoprotein b and fibrinogen. CONCLUSIONS: Detergent sclerosants affect the clotting mechanism by interfering with clotting factor activities, procoagulant phospholipids and platelet derived microparticles. STS has more anticoagulant activity than POL in high concentrations. Low concentration sclerosants demonstrate procoagulant activity.     

Burkholderia pseudomallei sequencing identifies genomic clades with distinct recombination,accessory, and epigenetic profiles

Burkholderia pseudomallei (Bp) is the causative agent of the infectious disease melioidosis. To investigate population diversity, recombination, and horizontal gene transfer in closely related Bp isolates, we performed whole-genome sequencing (WGS) on 106 clinical, animal, and environmental strains from a restricted Asian locale. Whole-genome phylogenies resolved multiple genomic clades of Bp, largely congruent with multilocus sequence typing (MLST). We discovered widespread recombination in the Bp core genome, involving hundreds of regions associated with multiple haplotypes. Highly recombinant regions exhibited functional enrichments that may contribute to virulence. We observed clade-specific patterns of recombination and accessory gene exchange, and provide evidence that this is likely due to ongoing recombination between clade members. Reciprocally, interclade exchanges were rarely observed, suggesting mechanisms restricting gene flow between clades. Interrogation of accessory elements revealed that each clade harbored a distinct complement of restriction-modification (RM) systems, predicted to cause clade-specific patterns of DNA methylation. Using methylome sequencing, we confirmed that representative strains from separate clades indeed exhibit distinct methylation profiles. Finally, using an E. coli system, we demonstrate that Bp RM systems can inhibit uptake of non-self DNA. Our data suggest that RM systems borne on mobile elements, besides preventing foreign DNA invasion, may also contribute to limiting exchanges of genetic material between individuals of the same species. Genomic clades may thus represent functional units of genetic isolation in Bp, modulating intraspecies genetic diversity.Burkholderia pseudomallei (Bp) is the causative agent of melioidosis, a serious infectious disease of humans and animals and a leading cause of community-acquired sepsis and pneumonia in endemic regions (Currie et al. 2010). Initially thought to be confined to Southeast Asia and Northern Australia, the prevalence of Bp appears to be spreading (Wiersinga et al. 2012), and Bp has been designated a biothreat select agent in the United States. Bp can persist in extreme environmental conditions and can infect several plant and animal hosts, including birds, dolphins, and humans (Wuthiekanun et al. 1995; Howard and Inglis 2003; Sprague and Neubauer 2004; Larsen et al. 2013). Treatment of clinical melioidosis is challenging because the bacterium is inherently resistant to many antibiotics, and Bp infections can persist in humans for more than a decade (Hayden et al. 2012; Wiersinga et al. 2012).The Bp genome comprises one of the largest and most complex bacterial genomes sequenced to date. Consisting of two large circular replicons (chromosomes) with a combined 7.2-Mb genome size (Holden et al. 2004), it contains a rich arsenal of genes related to virulence (e.g., Type III and Type VI secretion systems, polysaccharide biosynthesis clusters), metabolic pathways, and environmental adaptation (Wiersinga et al. 2012). Besides conserved regions, accessory genes on mobile elements and genomic islands may also contribute to phenotypic and clinical differences in microbial behavior (Currie et al. 2000; Sim et al. 2008). Analysis of the Bp genome has revealed previously unknown toxins and mechanisms of antibiotic resistance (Chantratita et al. 2011; Cruz-Migoni et al. 2011).Most large-scale studies of Bp genetic diversity to date have analyzed strains using multilocus sequence typing (MLST). These studies have suggested a high degree of genetic variability between Bp strains and related Burkholderia species (Cheng et al. 2008), and have shown that Bp strains belonging to different sequence types (STs) can often coexist in the same locale and sometimes even within the same sample (Pitt et al. 2007; Wuthiekanun et al. 2009). However, due to the limited number of genes analyzed by MLST, these studies cannot comment on the global proportion of genetic material shared between strains of different STs nor on the relative contribution of recombination, mutation, and horizontal gene transfer on intraspecies genetic diversity. Moreover, although previous studies have applied whole-genome sequencing (WGS) to study global patterns of Bp genetic heterogeneity and evolution, earlier Bp WGS reports have been confined to a limited number of isolates (10–12) derived from diverse geographical regions (Nandi et al. 2010), where geophysical barriers likely limit the propensity of the analyzed strains to exchange genetic material. To achieve a comprehensive understanding of genetic variation among closely related Bp strains, WGS analysis of much larger strain panels, ideally performed on strains isolated from a common region and belonging to the same (or closely related) ST groups, is required.In this study, we attempted to fill this important knowledge gap by performing WGS on 106 Bp strains drawn from a restricted Asian locale (Singapore and Malaysia). The WGS data, exceeding previous Bp WGS studies by 10-fold, enabled us to identify specific genomic clades of Bp, molecular features of Bp recombination at the whole-genome level, and accessory genome features contributing to recombination and horizontal gene transfer. We found a consistent pattern of genetic separation correlating with MLST, recombination haplotypes, shared accessory genes, and restriction modification (RM) systems. We provide evidence that restriction modification, beyond its role as defense against foreign DNA invasion, may have also partitioned the Bp species by restricting gene flow, resulting in the other observed correlations. Because RM systems are widely dispersed through the bacterial kingdom, it is possible that similar principles may apply to other bacterial species, implicating a potential role for epigenetic barriers as a driver of early incipient speciation.     

Clinical significance of the corrected orthogonal ECG (Frank) in comparison with conventional ECG for the detection of coronary heart disease

The clinical evidence of the two derivation systems is of the same value concerning the recognition of the coronary heart disease. Their information range does not significantly differ from each other statistically, when scale and vectorial parameters of the corrected orthogonal ECG after Frank are taken into consideration. But in individual cases clear differences may be present, such as the insufficient representation of localized precordial potential losses in the corrected orthogonal ECG. With the help of the discriminance analysis the high number of falsely negative findings of the ECG after resting can be reduced from about 45% to 15% to 20%. Within a diagnostic stage programme from the recognition and care of particularly endangered patients with coronary heart disease the corrected orthogonal ECG is suited, when a discriminance-analytic valuation with scale and vectorial parameters is made. In contrast to the conventional ECG it has the advantage that it is more rapidly and less expensively to be evaluated by computers.     

Paradoxical embolism, stroke and sclerotherapy

Sclerotherapy has been associated with 13 published cases of stroke since 1994. Four earlier reports implicated liquid sclerosants while nine recent cases have followed foam sclerotherapy. This adverse event represents a very rare complication of a very popular procedure. Ten of the 13 reported patients recovered completely with no long-term sequelae. A right-to-left shunt and in particular a patent foramen ovale (PFO) was the most consistent risk factor. Paradoxical gas emboli were observed in the brain-supplying or the intra-cranial arteries of five patients with an immediate onset of stroke after foam sclerotherapy. Paradoxical clot embolism was suspected in three patients with a delayed onset of stroke and concurrent venous thrombosis. In the remaining five cases, which included two cases with an immediate onset after liquid sclerotherapy, no specific cause was identified. Patients with a past history of cryptogenic stroke or a long life history of recurrent classic migraine attacks (with aura) have a higher risk of neurological adverse events and may benefit from preoperative screening and percutaneous closure of PFO.     

Biomechanical comparison of locked plating and spiral blade retrograde nailing of supracondylar femur fractures

Objective

Biomechanical comparison between locked plating and retrograde nailing of supracondylar femur fractures with simulated postoperative weight-bearing.

Methods

The Locking Condylar Plate (LCP) and Retrograde/Antegrade EX Femoral Nail (RAFN) were tested using 10 paired elderly cadaveric femurs, divided into Normal and Low Bone Mineral Density (BMD) groups, with a simulated AO/OTA type 33-A3 supracondylar femur fracture. Each specimen was subjected to 200,000 loading cycles in an attempt to simulate six weeks of postoperative recovery with full weight-bearing for an average individual. The construct's subsidence due to cyclic loading, and axial stiffness before and after the cyclic loading were measured and their correlation with BMD was studied. The two implants were compared in a paired study within each BMD group.

Results

LCP constructs showed higher axial stiffness compared to RAFN for both Normal and Low BMD groups (80% and 57%, respectively). After cyclic loading, axial stiffness of both constructs decreased by 20% and RAFN constructs resulted in twice as much subsidence (1.9 ± 0.6 mm). Two RAFN constructs with Low BMD failed after a few cycles whereas the matched pairs fixed with LCP failed after 70,000 cycles.

Conclusions

The RAFN constructs experienced greater subsidence and reduced axial stiffness compared to the LCP constructs. In Low BMD specimens, the RAFN constructs had a higher risk of failure.