Quorum-sensing linked RNA interference for dynamic metabolic pathway control in Saccharomyces cerevisiae.

Some of the most productive metabolic engineering strategies involve genetic modifications that cause severe metabolic burden on the host cell. Growth-limiting genetic modifications can be more effective if they are 'switched on' after a population growth phase has been completed. To address this problem we have engineered dynamic regulation using a previously developed synthetic quorum sensing circuit in Saccharomyces cerevisiae. The circuit autonomously triggers gene expression at a high population density, and was linked with an RNA interference module to enable target gene silencing. As a demonstration the circuit was used to control flux through the shikimate pathway for the production of para-hydroxybenzoic acid (PHBA). Dynamic RNA repression allowed gene knock-downs which were identified by elementary flux mode analysis as highly productive but with low biomass formation to be implemented after a population growth phase, resulting in the highest published PHBA titer in yeast (1.1mM).

Not so sweet: autoimmune diabetes mellitus on triple therapy for chronic hepatitis C infection.

BACKGROUND: Triple therapy with pegylated interferon, ribavirin and a protease inhibitor has proven efficacy in hepatitis C infection and is currently the standard of care. Interferon-based therapies have been, rarely, associated with the development of Type 1 diabetes mellitus, but few cases have yet been reported in triple therapy for hepatitis C. CASE REPORT: We describe a case of autoimmune Type 1 diabetes developing in a 23-year-old woman after initiation of triple therapy for chronic hepatitis C virus infection. The patient had the IL-28B gene polymorphism rs12979860 CT genotype, which is associated not only with antiviral therapy response but also with diabetes risk after liver transplantation for hepatitis C. CONCLUSION: Further studies are required to determine which individual characteristics may identify patients who are at risk of developing Type 1 diabetes when treated with interferon-based regimens for hepatitis C infection.

Improving pressure ulcer risk assessment and management using the Waterlow scale at a London teaching hospital.

OBJECTIVE: Pressure ulcers (PUs) cost the National Health Service (NHS) up to 4% of its health care expenditure. Arising from this are also clinical negligence claims, where inadequate risk assessment has been cited as one of the principal drawbacks in the prevention of PUs. This two-cycle audit aims to examine the consistency and accuracy of risk assessment of patients, and demonstrates how simple focused interventions can improve the quality of care provided. METHOD: The Waterlow pressure ulcer risk assessment tool was employed to assess inpatients during a 6-month period at a London teaching hospital. Patients were risk assessed, and examined to detect PUs and to determine the type of mattress. We compared our findings with clinical (nursing and medical) documentation. Interventions were made through questionnaires given to staff, educational sessions, presentations and posters addressing where improvements could be made in risk stratifying patients. A repeat audit was carried out 24 months later and the results from both cycles were compared. Statistical analysis was carried out using Fisher's exact and the Student's T-test. RESULTS: In total 100 in-patients were assessed in each cycle with a mean age of 71.4 years in cycle 1 and 70.1 years in cycle 2. A nursing Waterlow score was recorded for 81% of patients in cycle 1 and 100% in cycle 2 (p<0.05). In cycle 1, the average nursing score was significantly lower than that from the study (mean 13.7 versus 17.1, median 14.0 versus 18.0; p<0.05), but after intervention this had reduced to a minimal difference (mean 8.5 versus 9.0, median 8.0 versus 9.0, p=0.08). CONCLUSION: Nursing scores recorded in the notes were lower than the study scores in cycle 1, primarily from a failure to appropriately assess certain categories of the Waterlow scale. These differences reduced after focused education of staff. Our results suggest that targeted interventions tailored towards nursing and medical staff can result in improvements in the risk assessment for prevention and subsequent management of PUs. However, it also highlights the need for increased input from the entire multidisciplinary team in order to reduce the morbidity caused by PUs. DECLARATION OF INTEREST: The authors have no conflict of interest. No funding was received for this study.

New developments in the evolution and application of the WHO/IPCS framework on mode of action/species concordance analysis.

The World Health Organization/International Programme on Chemical Safety mode of action/human relevance framework has been updated to reflect the experience acquired in its application and extend its utility to emerging areas in toxicity testing and non-testing methods. The underlying principles have not changed, but the framework's scope has been extended to enable integration of information at different levels of biological organization and reflect evolving experience in a much broader range of potential applications. Mode of action/species concordance analysis can also inform hypothesis-based data generation and research priorities in support of risk assessment. The modified framework is incorporated within a roadmap, with feedback loops encouraging continuous refinement of fit-for-purpose testing strategies and risk assessment. Important in this construct is consideration of dose-response relationships and species concordance analysis in weight of evidence. The modified Bradford Hill considerations have been updated and additionally articulated to reflect increasing experience in application for cases where the toxicological outcome of chemical exposure is known. The modified framework can be used as originally intended, where the toxicological effects of chemical exposure are known, or in hypothesizing effects resulting from chemical exposure, using information on putative key events in established modes of action from appropriate in vitro or in silico systems and other lines of evidence. This modified mode of action framework and accompanying roadmap and case examples are expected to contribute to improving transparency in explicitly addressing weight of evidence considerations in mode of action/species concordance analysis based on both conventional data sources and evolving methods.

Transmission of hepatitis C virus to recipients of parenteral vitamin therapy in a primary care facility.

BACKGROUND AND OBJECTIVES: The Australian prevalence of hepatitis C virus (HCV) is approximately 1%, with the majority of cases acquired through injecting drug use. However, occasionally HCV infection occurs in healthcare settings. Three new HCV infections were identified amongst patients attending a general practice in Sydney, Australia, specialising in parenteral vitamin therapy. STUDY DESIGN: An investigation was conducted to identify the source of infection and mechanism of transmission. Molecular analysis was conducted by sequencing the HCV NS5A, Core and NS5B regions. RESULTS: Two sources were identified using molecular epidemiology - a genotype 3a case was the source for a case acquired in late 2004 and a genotype 1b case the source for one case acquired in late 2006 and another in early 2007. The common risk factor was parenteral vitamin C therapy. CONCLUSIONS: Inadequate infection control was apparent and likely to have resulted in blood contamination of the healthcare workers, their equipment, the clinic environment and parenteral medications. Molecular and clinical epidemiology clearly identified parenteral transmission of HCV, highlighting the risks of blood contamination of parenteral equipment and use of multi-dose flasks on more than one patient.

Production of bacteriocins by Streptococcus bovis strains from Australian ruminants.

AIMS: To examine the prevalence of bacteriocin production in Streptococcus bovis isolates from Australian ruminants and the feasibility of industrial production of bacteriocin. METHODS AND RESULTS: Streptococcus bovis strains were tested for production of bacteriocin-like inhibitory substances (BLIS) by antagonism assay against Lactococcus lactis. BLIS production was associated with source animal location (i.e. proximity of other bacteriocin-positive source animals) rather than ruminant species/breed or diet. One bacteriocin showing strong inhibitory activity (Sb15) was isolated and examined. Protein sequence, stability and activity spectrum of this bovicin were very similar to bovicin HC5. Production could be increased through serial culturing, and increased productivity could be partially maintained during cold storage of cultures. CONCLUSIONS: BLIS production is geographically widely distributed in Eastern Australia, and it appears that the bacteriocin(+) trait is maintained in animals at the same location. The HC5-like bacteriocin, originally identified in North America, is also found in Australia. Production of bacteriocin can be increased through serial culturing. SIGNIFICANCE AND IMPACT OF THE STUDY: The HC5-like bacteriocins appear to have a broad global distribution. Serial culturing may provide a route towards commercial manufacturing for use in industrial applications, and purified bacteriocin from S. bovis Sb15 could potentially be used to prevent food spoilage or as a feed additive to promote growth in ruminant species.

Late-phase, protein synthesis-dependent long-term potentiation in hippocampal CA1 pyramidal neurones with destabilized microtubule networks.

BACKGROUND AND PURPOSE: Protein synthesis-dependent late-long term potentiation (L-LTP) is an enduring form of synaptic plasticity that has been shown to rely on, at least partly, protein synthesis at synaptic and/or dendritic sites. Evidence suggests that somatic transcription of new mRNAs may provide a significant contribution to the availability of mRNAs at synaptic sites where they are made available for dendritic translation. Transport of mRNAs from somatic to dendritic sites might be expected to involve movement along a microtubule network. In this study we examined whether it was possible to maintain L-LTP in hippocampal slices with destabilized microtubule networks. EXPERIMENTAL APPROACH: Extracellular field excitatory postsynaptic potentials (fEPSPs) were recorded from rat hippocampal slices and following a period of baseline recording, stimuli were given that induced LTP. LTP was monitored for 5 h in both control slices and slices treated with vincristine to depolymerize tubulin. KEY RESULTS: L-LTP was induced and maintained in vincristine-treated slices. Four hours after tetanic stimulation fEPSPs were 196+/-19% of baseline values. The magnitude of potentiation was similar to that seen in untreated slices (175+/-15%). L-LTP in vincristine-treated slices was, however, not maintained in the presence of the protein synthesis inhibitor, rapamycin. Immunohistochemistry and confocal microscopy of vincristine-treated slices verified that the microtubule network had been destabilized. CONCLUSIONS AND IMPLICATIONS: Communication between somatic and synaptic sites through protein and/or mRNA trafficking via an intact microtubule network is not required for protein synthesis dependent L-LTP.

A synthetic xylanase as a novel reporter in plants.

Transient gene expression assays are often used to screen promoters before stable transformation. Current transient quantification methods have several problems, including a lack of reporter gene stability and expense. Here we report a synthetic, codon-optimised xylanase gene ( sXynA) as a reporter gene for quantitative transient analyses in plants. Azurine-crosslinked xylan (AZCL-xylan) was used as a substrate for assaying xylanase activity. The enzymatic nature of the protein allows for sensitive assays at the low levels of transgene protein found in transiently transformed tissue extracts. The xylanase (XYN) protein is stable, activity slopes are linear over long time periods and assays are cost-effective. Coupled with the GUS Plus reporter gene, the XYN reporter allows sensitive and accurate quantification of gene control sequences in transient expression systems.

Selectable marker-free transgenic barley producing a high level of cellulase (1,4-beta-glucanase) in developing grains.

The use of barley grains as bioreactors for high-level production of cellulase (1,4-beta-glucanase) was investigated. A hybrid cellulase gene, cel-hyb1, driven by the rice GluB-1 promoter was expressed specifically in developing endosperm. Codon usage optimisation of cel-hyb1 increased its expression in barley grains 527-fold and led to cellulase production of up to 1.5% of total grain protein. CEL-HYB1 enzyme in barley grains was highly stable during post-harvest storage. Selectable marker gene ( hph) was subsequently eliminated from transgenic lines through segregation of hph from synthetic cel-hyb1 ( syn.cel-hyb1) in T1 progeny, using a binary plasmid containing hph and syn.cel-hyb1 in separate T-DNAs. These data suggest that barley grains can potentially be used for the commercial production of cellulase.

Regression of gastric T cell lymphoma with eradication of Helicobacter pylori.

Helicobacter pylori is thought to be important in the pathogenesis of chronic active gastritis, peptic ulceration, gastric adenocarcinoma, and gastric B cell lymphoma of mucosa associated lymphoid tissue. The mechanism of evolution from chronic gastritis to monoclonal B cell proliferation is not known but is thought to be dependent on antigen specific T cells to H pylori and its products. Here, we report a case of gastric T cell lymphoma associated with chronic H pylori gastritis which regressed with eradication of the organism. This is the first report of a gastric T cell lymphoma regressing with H pylori eradication, and suggests a causal link between primary gastric T cell lymphoma and this organism.

Detecting cervical infection among family planning clients: difficulties at the primary health-care level in Indonesia.

In developing and testing an operational model for the integration of reproductive tract infection/sexually transmitted disease (RTI/STD) management into existing family planning (FP) services in Indonesia, this study allowed for assessment of disease prevalence and evaluation of diagnostic methods for detection of endocervicitis caused by chlamydial infection and/or gonorrhoea. Data were collected over 28 weeks in 1997 at 2 FP clinics in the low-income harbour neighbourhood of North Jakarta. Among 486 consenting female FP clients, prevalence of chlamydial infection was 9.3%, gonorrhoea 1.2%, trichomoniasis 4.5% and syphilis 0.8%. Clinically observed abnormal vaginal discharge, cervical inflammation and vaginal lesions/ulcers were all associated with cervical infection (P<0.05), but insufficiently sensitive (<60%). Clinical diagnosis for cervical infection had 48.8% sensitivity, 75.4% specificity, but only 18.3% positive predictive value (PPV). On-site Gram stains for gonorrhoea were 83.3% sensitive and 94.5% specific, but had only 16.1% PPV. Presence of mucopurulent cervicitis was only 39.6% sensitive for cervical STD, with PPV of only 16.3%. Development of an affordable and accurate detection tool for chlamydial infection remains the main obstacle to effective RTI/STD management in this population.

Burkitt's lymphoma associated with Helicobacter pylori.

The association between Helicobacter pylori infection and low grade mucosa-associated lymphoid tissue lymphoma is now widely accepted. In this report, we describe the concurrent development of Burkitt's lymphoma in the stomach of a 53-year-old male with perforated duodenal ulcer and positive H. pylori serology. The temporal relationship between these two events raises the possibility of a causal link between H. pylori infection and this lymphoproliferative disease. In describing this rare case of gastric Burkitt's lymphoma, we consider the evidence that supports this possibility.

Sulphoxidation and sulphation capacity in patients with primary biliary cirrhosis.

We have previously reported an association of impaired S-oxidation with primary biliary cirrhosis. In order to confirm and further define this relationship, we retested S-oxidation capacity via three metabolic pathways and sulphation capacity via a fourth pathway. Metabolism of S-carboxymethyl-L-cysteine is polymorphic -20% of healthy individuals being poor S-oxidisers. We found 26% with primary biliary cirrhosis were poor S-oxidisers, compared with 36% with other liver disease and 25% of healthy controls. Differences were not statistically significant. S-oxidation of ranitidine is dependent upon flavin mono-oxygenases. We showed a non-significant trend toward less S-oxide in primary biliary cirrhosis and other liver disease, compared with healthy controls, with no significant difference between disease groups. Conversion of cysteine to sulphate depends predominantly on cysteine dioxygenase. Impaired activity may be reflected by decreased plasma sulphate and elevated cysteine. We found that the plasma cysteine: sulphate ratio was significantly elevated not only in primary biliary cirrhosis (p < 0.0001), but also in other liver disease (p < 0.0001), compared with healthy individuals. Sulphation capacity was studied by analysing paracetamol metabolism. Paracetamol sulphate and sulphate: glucuronide ratio were reduced in primary biliary cirrhosis compared with normal individuals, (p < 0.05). A trend towards less sulphate in primary biliary cirrhosis compared other liver disease was not significant (p = 0.42). We conclude that although sulphation and some sulphoxidation pathways are impaired in primary biliary cirrhosis, we can currently find no evidence to substantiate the hypothesis that primary biliary cirrhosis is a disease specifically associated with poor S-oxidation, as assessed via these metabolic pathways.

Prevention of rebleeding from oesophageal varices: two-year follow up of a prospective controlled trial of propranolol in addition to sclerotherapy.

A prospective randomised trial comparing propranolol and sclerotherapy to sclerotherapy alone was conducted over a 2-year follow up in a district hospital setting of unselected patients. Rebleeding and survival were analysed. Thirty-nine patients were randomised to propranolol plus sclerotherapy and 34 to sclerotherapy alone. The two groups were clinically comparable. There was no significant difference in the cumulative percent of patients free of rebleeding; 54% of the sclerotherapy group rebled compared to 52% of the group treated with propranolol plus sclerotherapy (Hazard ratio 1.09 (0.54-2.22) and p = 0.81, NS). Two-year actuarial survival was also not significantly different, with 77% of the propanolol plus sclerotherapy group surviving, compared to 74% of sclerotherapy alone (Hazard ratio 1.08 (0.35-2.22) and p = 0.79, NS). The mean time to eradication of varices was not significantly different between the two groups (propranolol plus sclerotherapy 222 days, sclerotherapy alone 243 days), nor did the rate of variceal recurrence differ (72.7 vs 72 days). This study did not show long-term improvement in rebleeding or survival using propranolol in addition to a regular sclerotherapy programme.

Prospective, multicenter study of managing lower extremity venous ulcers.

Seventy patients with 90 venous ulcers were randomly assigned to hydrocolloid or conventional dressing and compression therapy at four study centers. The ulcers had been present for a mean of 47.8 in the control and 46.2 weeks in the treatment group and 42% of all patients had recurrent ulcers. Ulcers treated with hydrocolloid dressings reduced 71% and control treated wounds reduced 43% in area after 7.2 weeks of treatment. Thirty-four percent of all ulcers healed. Mean time to healing was 7 weeks for the hydrocolloid dressing group and 8 weeks for the control group. Most ulcers were less painful at final evaluation, but reduction in pain was more pronounced in hydrocolloid-dressed ulcers (p = 0.03). At baseline as well as during follow-up, significant differences between study centers were observed. Ulcers in patients in the United Kingdom were larger and less likely to heal (p = 0.001). Size of the ulcer at baseline was associated with treatment response and time to healing (p = 0.002). Percent reduction in ulcer area after 2 weeks was also correlated with treatment outcome (p = 0.004) and time to healing (p = 0.002). When all treatment outcome predictors were analyzed together, only percent reduction in area after 2 weeks remained statistically significant (p = 0.002), with percent reduction during the first 2 weeks of treatment > 30% predicting healing.

Development and isotype diversity of antibodies to inhalant and dietary antigens in childhood atopic eczema.

This study demonstrated that a major feature of childhood atopic eczema (AE) is the presence of serum IgG and IgE anti-house dust mite (anti-HDM) antibodies in almost all AE individuals. IgG anti-HDM antibodies, usually of the IgG1 isotype, became prevalent in AE children over the age of 4 years with the highest antibody levels in children in their early teens. In contrast, immunological sensitization to dietary antigens, notably milk and eggs, occurred in both AE children and age-matched non-atopic control children, and was often associated with IgG4 antibodies during early childhood. These became less prevalent with increasing age in control children but persisted in AE children, sometimes together with IgE antibodies. The later occurrence of anti-HDM antibodies in AE children could reflect immunological sensitization following inhalation of antigen, whereas sensitization to dietary antigens appears to be a natural event in early childhood.

Pruritus and cholestasis: therapeutic options.

The pathogenesis of pruritus of cholestasis remains unclear. Bile salts do not appear to be the sole prurogens in cholestasis. Histaminergic pathways may be involved, and central opiate receptor processes seem much more important than has previously been recognized. The therapeutic options for relief of cholestatic pruritus are summarized in Table 2. Resins such as cholestyramine are the first line of therapy. In cases where cholestyramine has failed, rifampicin and antihistamines may be beneficial. Opiate antagonists hold great potential if opioid withdrawal-like syndromes can be avoided. Ursodeoxycholic acid and methotrexate have an advantage in not only relieving pruritus but also potentially retarding disease progression in PBC and PSC, respectively, although this remains to be proved. Other agents such as EPO and SAMe remain experimental and require further study to clarify their effectiveness before they can be recommended.

Heat stroke following Rugby League football.

OBJECTIVE: To present a case of severe heat stroke after Rugby League football. CLINICAL FEATURES: A 29-year-old Rugby League forward with a mild infection of the upper respiratory tract collapsed while playing football in late March, when the ambient temperature was 24.1 degrees C and the relative humidity up to 73%. He was initially thought to have sustained a head injury and was markedly dehydrated. He suffered severe disseminated intravascular coagulation and gross neurological, renal and hepatic disturbances. INTERVENTION AND OUTCOME: He required repeated haemodialysis, assisted ventilation and supportive therapy and remained unconscious for 10 days. He then recovered fully. CONCLUSION: Heat stroke is potentially fatal and can be easily mistaken for head injury in contact sports. When players are dehydrated, have febrile illness and play in warm conditions, they may succumb to heat stroke.

Role of endoscopic retrograde cholangiopancreatography after orthotopic liver transplantation.

Twelve of 178 (7%) liver transplant patients underwent endoscopic retrograde cholangiopancreatography (ERCP) after transplantation. The indications for ERCP were persistent or late onset cholestasis, recurrent cholangitis, and suspected biliary leaks or strictures. The time between transplantation and ERCP ranged from 44 to 330 days (median 153 days). Biliary complications diagnosed by ERCP included biliary sludge in the form of casts, calculi, or debris (n = 7); bile leaks (n = 2); a biliary stricture (n = 1), and complete biliary obstruction (n = 1). One patient had a normal cholangiogram after transplantation. Biliary sludge was detected by ultrasound before ERCP in only one of six patients. Eight patients underwent endoscopic papillotomy, followed by clearance of biliary sludge in four and dilatation of a biliary stricture in one. Two patients bled after papillotomy but neither required surgical intervention. At a median follow up of 1.2 years (range 0.5-2.8 years), nine patients are well and three have died. ERCP provides both accurate diagnosis of biliary complications after liver transplantation and treatment that obviates the need for additional surgery in selected patients.