The cytoplasmic heritable determinant [PSI ؉ ] of the yeast Saccharomyces cerevisiae reflects the prion-like properties of the chromosome-encoded protein Sup35p. This protein is known to be an essential eukaryote polypeptide release factor, namely eRF3. In a [PSI ؉ ] background, the prion conformer of Sup35p forms large oligomers, which results in the intracellular depletion of functional release factor and hence inefficient translation termination. We have investigated the process by which the [ curing ͉ Sup35p ͉ cytoplasmic determinant R ecent biochemical evidence (1-5) has supported the hypothesis that the [PSI ϩ ] phenotype of Saccharomyces cerevisiae reflects the prion-like properties of the SUP35 gene product (6, 7). The essential chromosome-encoded protein Sup35p is known to be one of two eukaryote polypeptide release factors, namely eRF3 (8, 9). Sup35p associates with Sup45p (eRF1) in vivo to mediate translation termination (8). In vitro, Sup35p forms highly ordered fibers, whose appearance resembles that of fibrils formed by other amyloidogenic polypeptides (3,4 , a second prion determinant of S. cerevisiae (7). Other reagents including methanol, ethylene glycol, and hypertonic conditions, have been reported to exhibit curing properties; however, none of these cure with the neartotal efficiency of GuHCl (13,14).Two hypotheses have been proposed to account for the curing properties of GuHCl. First, the elimination of the prion might arise directly from the ability of GuHCl to denature proteins. However, the concentrations of GuHCl effective in curing [PSI ϩ ] are in the millimolar range, rather than the molar range typically required for the denaturation of proteins in vitro (13). Alternatively, GuHCl may actually promote the expression of an ancillary factor, namely the stress protein Hsp104p, which indirectly results in the reactivation of Sup35p and consequently the loss of the prion (15). Unlike other heat shock proteins, Hsp104p does not act to protect proteins against stress (i.e., heat denaturation), rather Hsp104p actively promotes the recovery of stress-denatured aggregated proteins by facilitating their refolding back into functional, native conformations (16,17). Overexpression of Hsp104p might lead to the total refolding of Sup35p from the aberrant prion conformation to its native structure, thereby mediating prion loss. To test both hypotheses, we have examined the kinetics of prion elimination upon growth in the presence of GuHCl and assessed the influence of stress on the curing process. Materials and MethodsStrain. The genotype of the strain used in this study was BSC783͞4a: , MATa. Growth Media. BSC783͞4a was grown at 30°C on 1 ⁄4YEPD solid medium [4% (wt͞vol) glucose, 1% (wt͞vol) Bacto-peptone, 0.25% (wt͞vol) yeast extract, 2% (wt͞vol) agar]. Most liquid cultures also were grown at 30°C in YEPD complete medium [2% (wt͞vol) glucose, 1% (wt͞vol) Bacto-peptone, 1% (wt͞vol) yeast extract], with or without 3 mM GuHCl. For studies using ethanol-supplemented media, strains were grown in f...

Purpose: Contemporary trauma resuscitation prioritizes control of bleeding and uses major haemorrhage protocols (MHPs) to prevent and treat coagulopathy. We aimed to determine whether augmenting MHPs with Viscoelastic Haemostatic Assays (VHA) would improve outcomes compared to Conventional Coagulation Tests (CCTs). Methods: This was a multi-centre, randomized controlled trial comparing outcomes in trauma patients who received empiric MHPs, augmented by either VHA or CCT-guided interventions. Primary outcome was the proportion of subjects who, at 24 h after injury, were alive and free of massive transfusion (10 or more red cell transfusions). Secondary outcomes included 28-day mortality. Pre-specified subgroups included patients with severe traumatic brain injury (TBI). Results: Of 396 patients in the intention to treat analysis, 201 were allocated to VHA and 195 to CCT-guided therapy. At 24 h, there was no difference in the proportion of patients who were alive and free of massive transfusion (VHA: 67%, CCT: 64%, OR 1.15, 95% CI 0.76-1.73). 28-day mortality was not different overall (VHA: 25%, CCT: 28%, OR 0.84, 95% CI 0.54-1.31), nor were there differences in other secondary outcomes or serious adverse events. In pre-specified subgroups, there were no differences in primary outcomes. In the pre-specified subgroup of 74 patients with TBI, 64% were alive and free of massive transfusion at 24 h compared to 46% in the CCT arm (OR 2.12, 95% CI 0.84-5.34). Conclusion: There was no difference in overall outcomes between VHA-and CCT-augmented-major haemorrhage protocols.

No abstract

The importance of receptor binding in the potent immunogenicity of Escherichia coli heat-labile enterotoxin B subunit (EtxB) was tested by comparing its immunogical properties with those of a receptor binding mutant, EtxB(G33D). Subcutaneous immunization of EtxB(G33D) resulted in 160-fold reduction in antibody titer compared with wild-type EtxB, whereas its oral delivery failed to provoke any detectable secretory or serum anti-B subunit responses. Moreover, the two proteins induced strikingly different effects on lymphocyte cultures in vitro. EtxB, in comparison with EtxB(G33D), caused an increase in the proportion of B cells, many ofwhich were activated (CD25+); the complete depletion of CD8+ T cells; an increase in the activation of CD4+ T cells; and an increase in interleukin 2 and a decrease in interferon 'y. These data indicate that EtxB exerts profound effects on immune cells, suggesting that its potent immunogenicity is dependent not only on efficient receptor-mediated uptake, but also on direct receptor-mediated immunomodulation of lymphocyte subsets.

We describe a systematic approach to define threshold parameters for ROTEM and TEG. These parameters were incorporated into algorithms to support data-driven adjustments of resuscitation with therapeutics, to optimize damage control resuscitation practice in trauma.

There is a high burden of trauma haemorrhage that affects all age groups. Research is required to understand the reasons for death after the first 24 h and barriers to timely transfusion support.

Recombinant adeno-associated virus (rAAV) vectors are a promising platform for in vivo gene therapy. The presence of neutralizing antibodies (Nab) against AAV capsids decreases cell transduction efficiency and is a common exclusion criterion for participation in clinical trials. Novel engineered capsids are being generated to improve gene delivery to the target cells and facilitate success of clinical trials; however, the prevalence of antibodies against such capsids remains largely unknown. We therefore assessed the seroprevalence of antibodies against a novel synthetic liver-tropic capsid AAV-LK03. We measured seroprevalence of immunoglobulin (Ig)G (i.e., neutralizing and nonneutralizing) antibodies and Nab to AAV-LK03 in a cohort of 323 UK patients (including 260 pediatric) and 52 juvenile rhesus macaques. We also performed comparative analysis of seroprevalence of Nab against wild-type AAV8 and AAV3B capsids. Overall IgG seroprevalence for AAV-LK03 was 39% in human samples. The titer increased with age. Prevalence of Nab was 23%, 35%, and 18% for AAV-LK03, AAV3B, and AAV8, respectively, with the lowest seroprevalence between 3 and 17 years of age for all serotypes. Presence of Nab against AAV-LK03 decreased from 36% in the youngest cohort (birth to 6 months) to 7% in older primary school-age children (9-11 years) and then progressively increased to 54% in late adulthood. Cross-reactivity between serotypes was >60%. Nab seroprevalence in macaques was 62%, 85%, and 40% for AAV-LK03, AAV3B, and AAV8, respectively. When planning for AAV gene therapy clinical trials, knowing the seropositivity of the target population is critical. In the population studied, AAV seroprevalence for AAV serotypes tested was low. However, high cross-reactivity between AAV serotypes remains a barrier for re-injection. Shifts in Nab seroprevalence during the first decade need to be confirmed by longitudinal studies. This possibility suggests that pediatric patients could respond differently to AAV therapy according to age. If late childhood is an ideal age window, intervention at an early age when maternal Nab levels are high may be challenging. Nab-positive children excluded from trials could be rescreened for eligibility at regular intervals because this status may change.

A high platelet or plasma to RBC ratio, and use of tranexamic acid were associated with a decreased need for massive transfusion and increased survival in injured patients with bleeding. Early normalization of coagulopathy was not seen for any transfusion ratio, or for use of tranexamic acid or fibrinogen products.