How do critical care staff respond to organisational challenge? A qualitative exploration into personality types and cognitive processing in critical care.

Critical care staff are frequently required to respond to stressful scenarios. The way staff counter organisational challenge may be influenced by their underlying personality type, preferred style of cognitive processing and previous clinical experience. Our objective was to explore the personality types of a sample of critical care workers, and the potential relationship of this with cognitive processing. This was achieved through a qualitative interview study in which participants were presented with difficult but realistic scenarios pertaining to staffing. Data on individual's personality were captured using the '16 Personality Factor' assessment, a tool that produces scores for 16 different elements of an individual's personality. The existence of perfectionist and pragmatic cognitive processing styles were identified as one theme emerging from a prior analysis of these interview transcripts. We aimed to validate this, explore our ability to categorise individuals into groups based upon their cognitive processing. We identified that some individuals strongly tended to either a perfectionist or pragmatic style of cognitive processing for the majority of their decisions; however most adapted their style of processing according to the nature of the decision. Overall participants generally demonstrated average scores for all 16 personality factors tested. However, we observed that some factors tended to higher scores than others, indicating a pattern within the personalities of our sample cohort. Whilst a small sample size, our data suggests that individuals working within the same critical care environment may have clear differences in their approach to problem solving as a consequence of both their personality type and preferred style of cognitive processing. Thus there may be individuals within this environment who would benefit from increased support to minimise their risk of cognitive dissonance and stress in times of challenge.

Knockdown of USP18 increases α 2a interferon signaling and induction of interferon-stimulating genes but does not increase antiviral activity in Huh7 cells.

The current standard of care for hepatitis C virus (HCV) patients is cotreatment with human alpha interferon (IFN-α) and ribavirin. The host factor USP18 functions to regulate the interferon signaling pathway by acting as an off-switch. In order to understand whether the inhibition of USP18 represents a valid target for the enhancement of interferon treatment for chronic viral diseases, we have used a wide range of RNA interference (RNAi) reagents to suppress USP18 gene expression in Huh7 cell lines. We demonstrate that a USP18 knockdown results in IFN-α2a signaling (measured by increased IFN-stimulated response element [ISRE] reporter gene activity, 2',5'-oligoadenylate synthetase [2-5 OAS] expression, and ISG15 induction) that is increased by ∼100-fold, whereas the antiviral (AV) potency in both the Huh7 HCV subgenomic replicon assay and the Huh7.5 HCV infectious virus assay increased by ∼3-fold. While the degree of the USP18 knockdown of USP18 elicited by the different RNAi reagents correlated with the enhancement of IFN-α2a signaling, it did not correlate with the enhancement of AV activity. The failure of increased IFN-α2a signaling to fully translate into increased AV potency was also observed for encephalomyocarditis virus (EMCV) assays using Huh7.5 cells. These data suggest that the IFN-mediated AV response in Huh7.5 cells has only a limited dependence on USP18 activity.

Selection, optimization, and pharmacokinetic properties of a novel, potent antiviral locked nucleic acid-based antisense oligomer targeting hepatitis C virus internal ribosome entry site.

We have screened 47 locked nucleic acid (LNA) antisense oligonucleotides (ASOs) targeting conserved (>95% homology) sequences in the hepatitis C virus (HCV) genome using the subgenomic HCV replicon assay and generated both antiviral (50% effective concentration [EC(50)]) and cytotoxic (50% cytotoxic concentration [CC(50)]) dose-response curves to allow measurement of the selectivity index (SI). This comprehensive approach has identified an LNA ASO with potent antiviral activity (EC(50) = 4 nM) and low cytotoxicity (CC(50) >880 nM) targeting the 25- to 40-nucleotide region (nt) of the HCV internal ribosome entry site (IRES) containing the distal and proximal miR-122 binding sites. LNA ASOs targeting previously known accessible regions of the IRES, namely, loop III and the initiation codon in loop IV, had poor SI values. We optimized the LNA ASO sequence by performing a 1-nucleotide walk through the 25- to 40-nt region and show that the boundaries for antiviral efficacy are extremely precise. Furthermore, we have optimized the format for the LNA ASO using different gapmer and mixomer patterns and show that RNase H is required for antiviral activity. We demonstrate that RNase H-refractory ASOs targeting the 25- to 40-nt region have no antiviral effect, revealing important regulatory features of the 25- to 40-nt region and suggesting that RNase H-refractory LNA ASOs can act as potential surrogates for proviral functions of miR-122. We confirm the antisense mechanism of action using mismatched LNA ASOs. Finally, we have performed pharmacokinetic experiments to demonstrate that the LNA ASOs have a very long half-life (>5 days) and attain hepatic maximum concentrations >100 times the concentration required for in vitro antiviral activity.

Kinetics of the homogeneous freezing of water.

Rates of homogeneous nucleation of ice in micrometre-sized water droplets are reported. Measurements were made using a new system in which droplets were supported on a hydrophobic substrate and their phase was monitored using optical microscopy as they were cooled at a controlled rate. Our nucleation rates are in agreement, given the quoted uncertainties, with the most recent literature data. However, the level of uncertainty in the rate of homogeneous freezing remains unacceptable given the importance of homogeneous nucleation to cloud formation in the Earth's atmosphere. We go on to use the most recent thermodynamic data for cubic ice (the metastable phase thought to nucleate from supercooled water) to estimate the interfacial energy of the cubic ice-supercooled water interface. We estimate a value of 20.8 +/- 1.2 mJ m(-2) in the temperature range 234.9-236.7 K.

A multidisciplinary method to map potential tuberculosis transmission 'hot spots' in high-burden communities.

BACKGROUND: Global control of the tuberculosis (TB) epidemic remains poor, especially in high-burden settings where ongoing transmission sustains the epidemic. In such settings, a significant amount of transmission takes place outside the household, and practical approaches to understanding transmission at community level are needed. OBJECTIVE: To identify and map potential TB transmission 'hot spots' across high-burden communities. SETTING AND DESIGN: Our method draws on data that qualitatively describe a high-burden community in Cape Town, South Africa. Established transmission principles are applied to grade the potential TB transmission risk posed by congregate settings in the community. Geographic information systems (GIS) technology then creates a visual map, locating potential transmission 'hot spots' in the community. RESULTS: Drinking places (shebeens), clinics and churches (often gatherings in confined homes) emerge as gathering places that potentially pose a high transmission risk, particularly if located in overcrowded and impoverished areas of the community. CONCLUSION: This proof-of-concept study demonstrates that combining qualitative techniques with GIS mapping may improve our understanding of potential TB transmission within a community and guide public health interventions to enhance TB control efforts.

Improved method for generating microarray probes using submicrogram amounts of total RNA.

The ability to identify disease-associated genes using microarray technology is dependent on isolating high-quality total RNA from the diseased tissue under study. However, it is not always possible to obtain large amounts of affected tissue from patients and therefore the quality and yield of RNA may be compromised. This protocol described a procedure for generating superior microarray probes by amplifying RNA sequences via successive rounds of in vitro transcription (IVT) reactions. This improved procedure utilizes a 9-mer primer to generate the IVT template, which is able to recapitulate the size ditribution of the original isolated RNA sample.

Effects of dietary restriction on appendicular bone in the SENCAR mouse.

Peptide hormones, cytokines, and growth factors regulate cellular metabolism by stimulating second messenger signal transduction cascades in target tissues. A mutation in the regulatory domain of protein kinase C (PKC) in SENCAR (sensitive to carcinogenesis) mice renders them extremely sensitive to diacylglycerol and phorbol esters, resulting in rapid growth, high free radical generation, carcinogenesis, and metabolic bone disease. Dietary restriction (DR) normalizes PKC and ameliorates adverse downstream effects, including carcinogenesis, in SENCAR mice. We hypothesized that DR sufficient to ameliorate carcinogenesis would prevent or delay the early onset of metabolic bone disease in SENCAR mice. Male mice were assigned to 1 of 4 feeding groups from 10 to 16 weeks of age (the critical period when metabolic bone disease develops): ad libitum (AL)-fed; AL antioxidant (0.07% thioproline)-fed; 40% DR; or 40% DR antioxidant-fed. Femoral bone mass was determined gravimetrically. Tibial total, cortical, and trabecular bone mineral density (BMD) were determined by quantitative computed tomography. Body weight, femoral bone mass, and tibial cortical BMD were lower in DR than in AL mice. However, tibial total and trabecular BMD were higher in DR than in AL mice. Serum calcitonin, the hormone that inhibits the osteoclastic bone resorption that is most notable in trabecular bone, was 2-fold higher in DR than in AL-fed mice. Dietary thioproline had no major effects. Thus, DR sufficient to ameliorate carcinogenesis in SENCAR mice did not prevent early-onset metabolic bone disease, but it had a beneficial effect on tibial trabecular BMD that occurred at the apparent expense of cortical BMD. DR in SENCAR mice was also associated with elevated serum calcitonin, which may inhibit osteoclastic resorption and account for trabecular bone conservation in this model. In conclusion, PKC or the downstream metabolic processes regulated by it appear to play previously unrecognized roles in the regulation of tibial trabecular BMD and serum calcitonin in SENCAR mice.

Empirical foundations for writing in prevention and psychotherapy: mental and physical health outcomes.

The use of writing, alone or in conjunction with traditional psychotherapy, has increased substantially in recent years. The most widespread use of writing has been for single-shot ad hoc purposes or to log behavior. The purpose of this review is to summarize a decade of research demonstrating the efficacy of writing about past traumatic experiences on mental and physical health outcomes. It is widely acknowledged in our culture that putting upsetting experiences into words can be healthy. Research from several domains indicates that talking with friends, confiding to a therapist, praying, and even writing about one's thoughts and feelings can be physically and mentally beneficial. This review highlights advances in written disclosure that determine some therapeutic outcomes. In addition, we attempt to explore the mechanisms that predict improved psychological and physical health. Finally, limitations of previous studies are highlighted, and suggestions for future research and application are made.

The ubiquitin-proteasome system and cellular proliferation and regulation in osteoblastic cells.

The 26S proteasome is the macromolecular assembly that mediates ATP- and ubiquitin-dependent extralysosomal intracellular protein degradation in eukaryotes. However, its contribution to the regulation of osteoblast proliferation and hormonal regulation remains poorly defined. Treating osteoblasts with MG-132 or lactacystin (membrane-permeable proteasome inhibitors) attenuates proliferation. Three proteasome activities (peptidylglutamyl-peptide bond hydrolase-, chymotrypsin-, and trypsin-like) were detected in osteoblasts. Catabolic doses of PTH stim-ulated these activities, and cotreatment with PTH and MG-132 blocked stimulation. The proteasome alpha- and beta-subunits, polyubiquitins, and large ubiquitin-protein conjugates were detected by Western blotting. A 90-min treatment with 10 nM PTH had no effect on the amount of proteasome alpha or beta subunit protein, but increased the relative amount of large ubiquitin-protein conjugates by 200%. MG-132 inhibited deubiquitination of large ubiquitin-protein conjugates. The protein kinase A inhibitor SQ22536 blocked much of the PTH-induced stimulation of MCP activities, while dibutyryl cAMP stimulated it, suggesting that protein kinase A-dependent phosphorylation is important in PTH stimulation of proteasome activities. In conclusion, the ubiquitin-proteasome system is essential for osteoblast proliferation under control and PTH-treated conditions. PTH mediates its metabolic effects on the osteoblast, in part, by enhancing ubiquitinylation of protein substrates and stimulating three major proteasome activities by a cAMP-dependent mechanism.

Ro 09-2210 exhibits potent anti-proliferative effects on activated T cells by selectively blocking MKK activity.

By using high throughput screening of microbial broths, we have identified a compound, designated Ro 09-2210, which is able to block anti-CD3 induced peripheral blood T cell activation with an IC50 = 40 nM. Ro 09-2210 was also able to block antigen-induced IL-2 secretion with an IC50 = 30 nM, but was considerably less potent at blocking Ca2+ flux stimulated by anti-CD3 treatment. To determine the mechanism of action of Ro 09-2210, we set up a transient expression system in Jurkat T cells using a variety of reporter gene constructs and showed effective inhibition of phorbol ester/ionomycin-induced NF-AT activation and anti-CD3 induced NF-AT with IC50 = 7.7 and 10 nM, respectively. Ro 09-2210 was also able to inhibit phorbol ester/ionomycin-induced activation of AP1 with IC50 = <10 nM. We further showed that Ro 09-2210 was unable to inhibit c-jun induced expression of AP1-dependent reporter constructs (IC50 > 500 nM), but was able to potently inhibit ras-induced AP1 activation (IC50 = 20 nM). This suggested that Ro 09-2210 was inhibiting an activator of AP-1 which was upstream of c-jun and downstream of ras signaling. To investigate further, we then purified a number of different kinases, including PKC, PhK, ZAP-70, ERK, and MEK 1 (a MKK), and showed that Ro 09-2210 was a selective inhibitor of MEK1 in vitro (IC50 = 59 nM).

E64d, a membrane-permeable cysteine protease inhibitor, attenuates the effects of parathyroid hormone on osteoblasts in vitro.

Parathyroid hormone (PTH) activates calpains I and II (calcium-activated papain-like proteases) and stimulates the synthesis and secretion of cathepsin B (a lysosomal cysteine protease) in osteoblastic cells. Anabolic doses of PTH also stimulate osteoprogenitor cell proliferation and differentiation into mature, fully functional osteoblasts capable of elaborating bone matrix, whereas catabolic doses of PTH stimulate calcium mobilization and matrix turnover. Previous investigations in other cell types have demonstrated that calcium-activated calpains play a major role in regulating proliferation and differentiation by catalyzing limited regulatory proteolysis of nuclear proteins, transcription factors, and enzymes. We tested the hypothesis that inhibition of intracellular cysteine proteases such as the calpains will ablate PTH-mediated osteoblast proliferation and differentiation, two fundamental indices of bone anabolism. A brief preincubation with the membrane-permeable, irreversible cysteine protease inhibitor E64d (10 micrograms/mL) before short-term PTH treatment blunted PTH-induced cell proliferation in subconfluent cultures and also attenuated proliferation and inhibited differentiation in longer-term confluent cultures. This confirms the hypothesis that cysteine proteases such as the calpains are important in mediating the proliferative and prodifferentiating or anabolic effects of PTH on MC3T3-E1 cells in culture. Immunofluorescent localization demonstrated that calpain I, calpain II, and calpastatin (the endogenous calpain inhibitor) are abundant and widely distributed within actively proliferating MC3T3-E1 preosteoblasts. Since the calpains are active and stable at neutral intracellular pH levels in osteoblasts, whereas cathepsins are not, our results support a role for these calcium-activated regulatory proteases in mediating the anabolic effects of PTH in bone.

Effect of low dietary calcium on bone metabolism in the SENCAR mouse.

The SENCAR (sensitive to carcinogenesis) mouse is a unique tool for investigating the interaction between a specific defect in intracellular signaling, dietary calcium, and metabolic bone disease. The SENCAR mouse was developed by selective breeding for enhanced sensitivity to two-stage carcinogenesis. Its major genetic defect, which renders it exquisitely sensitive to stimulation with diacylglycerol or phorbol esters, is in the regulatory domain of protein kinase C, one of the primary intracellular mediators of hormonal effects. At sexual maturity, SENCAR mice are large and have big bones, but our previous pharmacokinetic studies showed that they accumulate less calcium under normal conditions and lose more calcium under adverse conditions than do other, standard strains of mice. To histologically define the effect of low dietary calcium on bone metabolism, we performed histomorphometric analysis of tetracycline-labeled sections of femoral bone from male SENCAR mice maintained on calcium-sufficient and calcium-deficient diets during the critical period from 10 to 14 weeks of age. The bone volume, absolute osteoid volume, and mineral apposition rate were lower at 14 than at 10 weeks of age in SENCAR mice fed 0.02 or 0.6% calcium diets. Calcium deficiency increased the architectural disarray and the probability of observing focal discontinuities in the growth plate. Thus, characteristic features of impaired bone metabolism (low bone volume and apposition rate) develop early in SENCAR mice and are exacerbated by low dietary calcium. Detailed examinations of the histology and biochemistry of SENCAR mouse bone will provide insights into the mechanisms by which specific defects in the signal transduction of protein kinase C contribute to impaired bone metabolism.

The calpain-calpastatin system and cellular proliferation and differentiation in rodent osteoblastic cells.

The calpain-calpastatin system, which consists of calpains I and II (two ubiquitously distributed calcium-activated papain-like cysteine proteases), as well as calpastatin (the endogenous calpain inhibitor), plays an important role in cell proliferation and differentiation in many tissues. However, its contribution to the regulation of osteoprogenitor or pluripotent stem cell proliferation and differentiation into osteoblasts remains poorly defined. In these studies, rat pluripotent mesodermal cells (ROB-C26) and mouse MC3T3-E1 preosteoblasts were induced to differentiate into osteoblasts by long-term culture or in response to bone morphogenetic protein (BMP). The occurrence and distribution of calpain-calpastatin system proteins were determined by immunofluorescent microscopy, measurement of calcium-dependent proteolytic activity, and Western blotting. Treatment of intact MC3T3-E1 cells with an irreversible, membrane-permeable cysteine protease inhibitor attenuated proliferation and alkaline phosphatase upregulation under differentiation-enhancing conditions. Calpain II activity increased during differentiation of MC3T3-E1 cells in postconfluent culture. When ROB-C26 cells were maintained in long-term culture, neutral protease, calpain I, and calpain II activities increased 2- to 3-fold in the absence of BMP. In the presence of partially purified native BMP, neutral protease and calpain I activities also increased similarly, but calpain II activity increased by 10-fold in 3 days. The maximal increase in alkaline phosphatase occurred 4 to 11 days after the calpain II activity had peaked. Induction of differentiation in long-term MC3T3-E1 cultures was associated with higher calpain II and 70- and 110-kDa calpastatin protein levels and lower 17-kDa calpastatin degradation product levels. In conclusion, cysteine protease activity is essential for preosteoblastic proliferation and differentiation. The calpain-calpastatin system is regulated during osteoprogenitor proliferation and differentiation, as it is in other cells, and bone morphogenetic protein is a specific regulator of calpain II.

Divergent regulation by growth factors and cytokines of 95 kDa and 72 kDa gelatinases and tissue inhibitors or metalloproteinases-1, -2, and -3 in rabbit aortic smooth muscle cells.

The migration and proliferation of vascular smooth muscle cells (SMCs) during neointima formation in atherosclerosis and angioplasty restenosis is mediated by certain growth factors and cytokines, one action of which may be to promote basement-membrane degradation. To test this hypothesis further, the effects of such growth factors and cytokines on the synthesis of two basement-membrane-degrading metalloproteinases, namely the 72 kDa gelatinase (MMP-2, gelatinase A) and the 95 kDa gelatinase (MMP-9, gelatinase B) and three tissue inhibitors of metalloproteinases (TIMPs) was studied in primary cultured rabbit aortic SMCs. Expression of the 95 kDa gelatinase was increased by phorbol myristate acetate, foetal calf serum, thrombin and interleukin-1alpha (IL-1alpha); platelet-derived growth factor (PDGF) BB alone had no effect but acted synergistically with IL-1alpha. A selective protein kinase C inhibitor, Ro 31-8220, abolished induction of the 95 kDa gelatinase. In contrast, none of the agents tested modulated the synthesis of the 72 kDa gelatinase. We conclude that maximal up-regulation of 95 kDa gelatinase expression requires the concerted action of growth factors and inflammatory cytokines mediated, in part, by a protein kinase C-dependent pathway. TIMP-1 and TIMP-2 were highly expressed, and their synthesis was not affected by growth factors or cytokines. Expression of TIMP-3 mRNAs was, however, increased by PDGF and transforming growth factor beta, especially in combination. Divergent regulation of gelatinase and TIMP expression implies that either net synthesis or net degradation of basement membrane can be mediated by appropriate combinations of growth factors and cytokines.

Parathyroid hormone-induced retraction of MC3T3-E1 osteoblastic cells is attenuated by the calpain inhibitor N-Ac-Leu-Leu-norleucinal.

Parathyroid hormone (PTH) binding to its osteoblastic receptors stimulates cytoplasmic retraction within minutes. We hypothesized that the calpains (calcium-activated papain-like enzymes) contribute to PTH-induced osteoblastic retraction by catalyzing regulatory hydrolysis of cytoskeletal structural proteins or enzymes important in cytokinesis. N-Ac-Leu-Leu-norleucinal (ALLN), a reversible calpain inhibitor, was tested for its ability to inhibit PTH-induced retraction in murine MC3T3-E1 osteoblastic cells. ALLN inhibited PTH-induced retraction for 30 minutes in cells cultured on polystyrene cultureware or gelatin-coated glass cover slips, supporting the hypothesis that PTH-induced activation of the calpains contributes to short-term changes in MC3T3-E1 cell shape. Inhibition of PTH-induced retraction occurred on two substrata, suggesting that interactions between the extracellular matrix and cell surface proteins are not the sole determinants of morphology. Intracellular events, such as hydrolysis of focal adherens junction proteins on the cytoplasmic face of the plasma membrane, may contribute to PTH-induced retraction.

Protein kinase C is not a downstream effector of p21ras in activated T cells.

The aim of this present study was to investigate the role of protein kinase C (PKC), downstream of p21ras, in activating interleukin-2 (IL-2) gene expression. It has been reported that PKC is an effector of p21ras in T cells. Data is presented, using the potent and selective PKC inhibitor Ro 31-8425 and transient expression of a constitutively active ras mutant, which clearly shows that PKC is not downstream of p21ras in the induction of NF-AT and AP-1 transcriptional activity and in the expression of IL-2 in human Jurkat T cells. Reporter gene experiments demonstrated that NF-kappa B transcriptional activity is not affected by expression of activated p21ras. The signaling pathways involving PKC activation, calcium mobilization and ras activation combine to provide the necessary components for production of IL-2 during T cell activation.

PTH-mediated osteoblast retraction: possible participation of the calpain pathway.

Parathyroid hormone (PTH)-induced osteoblast retraction may play a pivotal role a role in bone resorption by providing osteoclasts direct access to mineralized bone surface. We have been working on the hypothesis that the calpains participate in this retractile response through a calcium-dependent process. We have first demonstrated the presence of calpain activities in MC3T3-E1 osteoblastic cells and that these activities can be stimulated by PTH. Second, we have demonstrated that the PTH-induced osteoblast retraction is dramatically attenuated by two different cysteine protease inhibitors. Finally, initial immunofluorescent cytochemical studies suggest that this PTH-induced osteoblastic retraction is mediated through a calpain-dependent, proteolytic modification of the cytoskeletal organization.

PTH and PTH-rP elicit dissimilar retractile responses in murine MC3T3-E1 osteoblasts.

Parathyroid hormone (PTH) and PTH-related peptide (PTH-rP) bind to a common receptor and initiate second-messenger cascades that stimulate bone turnover and hypercalcemia. However, PTH is more potent than PTH-rP in inducing bone resorption and coupled bone metabolism in intact tissue, suggesting that these proteins elicit dissimilar postreceptor responses. We compared the effects of PTH and PTH-rP on osteoblastic retraction, an early event that must occur before the osteoclast can achieve access to the underlying bone mineral and begin resorption. MC3T3-E1 mouse osteoblasts were incubated in vehicle or 4.8 nM PTH or PTH-rP with or without 1 mM dibutyryl cAMP (Bt2cAMP). Morphologic changes were observed from 0 to 120 min. PTH caused marked retraction within minutes, which was not enhanced by Bt2cAMP. PTH-rP or Bt2cAMP induced slower, more modest retraction than PTH. The combined effect of PTH-rP plus Bt2cAMP was greater than that of PTH-rP, but less than that of PTH. PTH-rP and PTH had similar effects on cAMP generation. Thus, compared to PTH, PTH-rP induces less osteoblastic retractile response, exposing less bone surface to osteoclastic resorption. This may account for its lower hypercalcemic potency in vivo and contribute to its relative inability to stimulate coupled bone resorption and formation.