Engineering pyruvate decarboxylase-mediated ethanol production in the thermophilic host Geobacillus thermoglucosidasius.

This study reports the expression, purification, and kinetic characterization of a pyruvate decarboxylase (PDC) from Gluconobacter oxydans. Kinetic analyses showed the enzyme to have high affinity for pyruvate (120 µM at pH 5), high catalytic efficiency (4.75 × 10(5) M(-1) s(-1) at pH 5), a pHopt of approximately 4.5 and an in vitro temperature optimum at approximately 55 °C. Due to in vitro thermostablity (approximately 40 % enzyme activity retained after 30 min at 65 °C), this PDC was considered to be a suitable candidate for heterologous expression in the thermophile Geobacillus thermoglucosidasius for ethanol production. Initial studies using a variety of methods failed to detect activity at any growth temperature (45-55 °C). However, the application of codon harmonization (i.e., mimicry of the heterogeneous host's transcription and translational rhythm) yielded a protein that was fully functional in the thermophilic strain at 45 °C (as determined by enzyme activity, Western blot, mRNA detection, and ethanol productivity). Here, we describe the first successful expression of PDC in a true thermophile. Yields as high as 0.35 ± 0.04 g/g ethanol per gram of glucose consumed were detected, highly competitive to those reported in ethanologenic thermophilic mutants. Although activities could not be detected at temperatures approaching the growth optimum for the strain, this study highlights the possibility that previously unsuccessful expression of pdcs in Geobacillus spp. may be the result of ineffective transcription/translation coupling.

Genetic tool development underpins recent advances in thermophilic whole-cell biocatalysts.

The environmental value of sustainably producing bioproducts from biomass is now widely appreciated, with a primary target being the economic production of fuels such as bioethanol from lignocellulose. The application of thermophilic prokaryotes is a rapidly developing niche in this field, driven by their known catabolic versatility with lignocellulose-derived carbohydrates. Fundamental to the success of this work has been the development of reliable genetic and molecular systems. These technical tools are now available to assist in the development of other (hyper)thermophilic strains with diverse phenotypes such as hemicellulolytic and cellulolytic properties, branched chain alcohol production and other 'valuable bioproduct' synthetic capabilities. Here we present an insight into the historical limitations, recent developments and current status of a number of genetic systems for thermophiles. We also highlight the value of reliable genetic methods for increasing our knowledge of thermophile physiology. We argue that the development of robust genetic systems is paramount in the evolution of future thermophilic based bioprocesses and make suggestions for future approaches and genetic targets that will facilitate this process.

Primary health eye care knowledge among general practitioners working in the Cape Town metropole

Aim: The main purpose of this study was to determine whether general practitioners (GPs) in the Cape Town metropole have sufficient knowledge to diagnose and treat primary care ophthalmic conditions correctly; and to assess their own perceptions of their levels of knowledge. Secondary objectives included identifying the need for courses to improve the ophthalmic knowledge of GPs and assessing whether there is a need to revise the undergraduate curriculum in ophthalmology in general. Method: A cross-sectional survey was done. A questionnaire of 10 primary care level ophthalmology questions; including a self-assessment section; was sent to each of 140 randomly chosen GPs in Cape Town. Results: A response rate of 79.2was obtained. Respondents included graduates from all eight medical schools in South Africa. Most of the responding GPs were practising for more than 10 years (78.2). The mean test score was 52.5(standard deviation [SD]: 22.2). The mean self-rating was 51.9(SD: 14.5). There was no statistically significant difference between the test score and the self-rating score (p = 0.5840). Responding GPs felt that there is a need for ophthalmology up-skilling courses and 99.9of them would attend such courses. Also; 82of GPs felt that primary care doctors; not optometrists; should deliver primary eye care. Conclusion: GPs appear to lack sufficient knowledge to manage primary health eye care problems; presumably due to a lack of adequate training in the field. Clinical up-skilling courses are needed to improve core knowledge in ophthalmology

Conjunctival Necrosis due to Subconjunctival Methylprednisolone (Depo-Medrol™) Acetate Injection.

We report a case of conjunctival necrosis due to subconjunctival methylprednisolone (Depo-Medrol™) acetate injection after phacoemulsification surgery. This case report highlights a serious complication of the inadvertent use of methylprednisolone as a subconjunctival agent. To report a case of conjunctival necrosis due to subconjunctival methylprednisolone (Depo-Medrol™) acetate injection after phacoemulsification. Case report a single case presenting to a tertiary ophthalmic unit. An 82-year-old patient underwent uncomplicated phacoemulsification in the right eye. Postoperatively, she was given a subconjunctival injection of methylprednisolone. Two weeks later, she presented with a painful ulcerated lesion of the conjunctiva proximal to the injection site. The ulcerated lesion was surgically excised and she made a complete recovery. In this reported case, methylprednisolone was used in error with significant resultant morbidity. This preparation is not registered for the off label use in ophthalmology, and this case report highlights the danger of its inadvertent use as a subconjuctival agent.

Phylogenetic relationships, character evolution and biogeography of southern African members of Zygophyllum (Zygophyllaceae) based on three plastid regions.

The plastid coding rbcL and non-coding trnLF regions of 53 of 55 southern African Zygophyllum species were sequenced and used to evaluate the phylogenetic relationships within the southern African representatives of the genus. Published sequences of the same gene regions of Australian, Asian and North African Zygophyllum species were included to assess the relationships of the species from these regions to the southern African species. The addition of Z. stapffii from Namibia, found to be conspecific with Z. orbiculatum from Angola, lead to a greatly resolved tree. The molecular results were largely congruent with a recent sectional classification of the southern African species and supported their subdivision into subgenera Agrophyllum and Zygophyllum. Reconstruction of the character evolution of capsule dehiscence, seed attachment and seed mucilage showed that these characters allowed a division of southern African species into the two subgenera but that this could not be applied to species occurring elsewhere. Other morphological characters were found to vary and unique character combinations, rather than unique characters, were found to be of systematic value in sectional delimitation. The study suggests that repeated radiations from the horn of Africa to southern Africa and Asia and back lead to the present distribution of the taxa in the subfamily Zygophylloideae. Although this study supports some of the recent taxonomic changes in the group, the unresolved relationships between the proposed genera Tetraena and Roepera and those retained as Zygophyllum species suggest that changes to the taxonomy may have been premature.

Indications of limited altered gene expression in Pinus sylvestris trees from the Chernobyl region.

To evaluate the consequences of irradiation on the vegetation of the Chernobyl region, gene expression was compared in morphologically normal and dwarf needles from the same Pinus sylvestris trees in a region where the absorbed dose was 3-5 Gy. To compare the levels of gene expression, arrays consisting of 373 Pinus taeda cDNAs were hybridized with labeled cDNA derived from normal and dwarf needles of P. sylvestris. Twelve genes were significantly (P<0.01) up- or down-regulated between normal and dwarf needles for all five trees taken together. Five of these, related to stress or development, were up- or down-regulated 1.25-1.7-fold in the dwarf needles. There were no significant differences in (137)Cs content in the normal and dwarf needles, or in elongation growth rate of seedlings raised under controlled conditions from seed derived from trees in the region that had received a radiation dose over the range 2-12 Gy.

Gene expression during formation of earlywood and latewood in loblolly pine: expression profiles of 350 genes.

The natural variability of wood formation in trees affords opportunities to correlate transcript profiles with the resulting wood properties. We have used cDNA microarrays to study transcript abundance in developing secondary xylem of loblolly pine (Pinus taeda) over a growing season. The cDNAs were selected from a collection of 75 000 ESTs that have been sequenced and annotated (http://web.ahc.umn.edu/biodata/nsfpine/). Cell wall thickness and climatic data were related to earlywood and latewood formation at different time points during the growing season. Seventy-one ESTs showed preferential expression in earlywood or latewood, including 23 genes with no significant similarity to genes in GenBank. Seven genes involved in lignin synthesis were preferentially expressed in latewood. The studies have provided initial insights into the variation of expression patterns of some of the genes related to the wood formation process.

Interpretations, perspectives and intentions in surrogate motherhood.

In this paper we examine the questions "What does it mean to be a surrogate mother?" and "What would be an appropriate perspective for a surrogate mother to have on her pregnancy?" In response to the objection that such contracts are alienating or dehumanising since they require women to suppress their evolving perspective on their pregnancies, liberal supporters of surrogate motherhood argue that the freedom to contract includes the freedom to enter a contract to bear a child for an infertile couple. After entering the contract the surrogate may not be free to interpret her pregnancy as that of a non-surrogate mother, but there is more than one appropriate way of interpreting one's pregnancy. To restrict or ban surrogacy contracts would be to prohibit women from making other particular interpretations of their pregnancies they may wish to make, requiring them to live up to a culturally constituted image of ideal motherhood. We examine three interpretations of a "surrogate pregnancy" that are implicit in the views and arguments put forward by ethicists, surrogacy agencies, and surrogate mothers themselves. We hope to show that our concern in this regard goes beyond the view that surrogacy contracts deny or suppress the natural, instinctive or conventional interpretation of pregnancy.

Intensive short course chemotherapy in the management of tuberculous meningitis.

SETTING: Short course chemotherapy for tuberculous meningitis (TBM) is advocated by several groups, but relatively few children have been so treated and followed up. METHODS: A prospective, observational study of isoniazid (INH), rifampicin (RMP) and ethionamide (ETH) in a dosage of 20 mg/kg, and pyrazinamide (PZA) 40 mg/kg, all given once daily in hospital for 6 months. Surviving children were followed up for a year after discharge. RESULTS: Ninety five children, 39 (41%) at stage III, 52 (55%) at stage II and 4 (4%) at stage I TBM were studied. Ten (26%) at stage III and 3 (6%) at stage II died before completion of therapy. Five surviving children (6%) moved on discharge and were untraceable; seven children (9%) were lost during follow up and three were inadvertently restarted on antituberculosis therapy. Two children with severe stage III disease died after discharge. One child experienced a probable disease recrudescence 1 month after discharge. Eighteen children (20%) developed a mildly elevated serum bilirubin concentration during the first month of treatment. In five of these children INH, RMP, ETH and PZA were stopped and streptomycin (SM) and ethambutol substituted. In all cases the original treatment was restarted without incident. One child developed overt jaundice after 5 months of treatment due to hepatitis A infection. CONCLUSIONS: Our experience suggests that young children with TBM can be safely treated for 6 months with high doses of antituberculosis agents without overt hepatotoxicity and with a low risk of relapse.

Effect of corticosteroids on intracranial pressure, computed tomographic findings, and clinical outcome in young children with tuberculous meningitis.

OBJECTIVE: To study the effect of highdose prednisone on intracranial pressure (ICP), cranial computed tomographic (CT) findings, and clinical outcome in young children with moderate to severe tuberculous meningitis (TBM). STUDY DESIGN: Prospective, controlled, randomized study. METHODS: Continuous lumbar, cerebrospinal fluid pressure monitoring and contrasted CT scanning were performed in 141 consecutive children with TBM at admission. All children were then randomly allocated to a nonsteroid group (71 children) or a steroid group (70 children) who received prednisone (first 16 children, 2 mg/kg per day; next 54 children, 4 mg/kg per day) for the first month of treatment. ICP monitoring and CT scanning were repeated regularly, and clinical outcome was assessed after 6 months of antituberculosis treatment. RESULTS: No statistically significant difference in ICP or the degree of hydrocephalus (as demonstrated by CT scan) was found between the steroid and nonsteroid groups after the first month of treatment. Basal ganglia infarcts developed in 16% of children in the steroid group and 24% in the nonsteroid group during the first month of treatment. Neither this incidence nor the eventual size of infarcts present at admission differed significantly between the two treatment groups. Single or multiple tuberculomas were seen on the first CT scans of 7 children (5%), whereas tuberculomas developed in 11 children (8%) at treatment. Both the response of the tuberculomas to treatment and the incidence of new tuberculomas were significantly improved by steroid therapy. Basal enhancement was also significantly less in the steroid group after 1 month of treatment. Steroids lowered mortality in stage III TBM significantly. Similarly, more surviving children in the steroid group had IQs of greater than 75 than did the those in the nonsteroid group. No significant difference was found in the incidence of motor deficit, blindness, or deafness. CONCLUSIONS: Corticosteroids significantly improved the survival rate and intellectual outcome of children with TBM. Enhanced resolution of the basal exudate and tuberculomas by steroids was shown by serial CT scanning. Corticosteroids did not affect ICP or the incidence of basal ganglia infarction significantly.

The ethics of surrogacy: women's reproductive labour.

The aim of this article is to establish whether there is anything intrinsically immoral about surrogacy arrangements from the perspective of the surrogate mother herself. Specific attention is paid to the claim that surrogacy is similar to prostitution in that it reduces women's reproductive labour to a form of alienated and/or dehumanized labour.

Serial CT scanning in childhood tuberculous meningitis: prognostic features in 198 cases.

Serial cranial computed tomographic (CT) scanning and intracranial pressure monitoring were performed on 198 children with stage II and III tuberculous meningitis. The aims of the study were to document the course of tuberculous hydrocephalus during medical and surgical treatment, as well as the prognostic significance of parenchymal changes in the brain as demonstrated by CT. Lumbar cerebrospinal fluid pressure was monitored continuously for a 1-hour period in all patients on admission and at weekly intervals in patients with communicating hydrocephalus for the 1st month of treatment. Cranial CT scanning was done on admission and repeated in survivors after 1 month and again after 6 months of antituberculous therapy. The raised intracranial pressure of 112 children with communicating hydrocephalus, as demonstrated by air-encephalography, was treated medically (with daily acetazolamide and furosemide) for 1 month. Thirty-one children with noncommunicating hydrocephalus were referred for immediate ventriculoperitoneal shunting. No significant difference was found in the eventual ventricular size or clinical outcome between the two treatment groups. Lumbar cerebrospinal fluid pressure changes in the children with communicating hydrocephalus closely followed changes in the degree of hydrocephalus during the course of treatment. The main cause of permanent neurologic disability was basal ganglia infarction, which occurred unilaterally in 21% and bilaterally in 10% of patients on admission and developed in a further 22% of children during treatment. A prominent subarachnoid space, which was seen on the CT scan of 36% of patients after the 1st month of treatment and which reverted to normal, probably relates to the poor nutritional state of these patients on admission.(ABSTRACT TRUNCATED AT 250 WORDS)

BCG vaccination status of children with tuberculous meningitis and the use of unsupervised isoniazid prophylaxis.

From 1985 to 1992, 193 children with tuberculous meningitis (TBM) with a median age of 26 months were admitted to the Department of Paediatrics and Child Health, Tygerberg Hospital. Of these children 143 (74%) were documented to have received BCG, either by reference to 'Road to Health' cards or by contact with local authority clinic staff. In a further 18 children a BCG scar was visible. Therefore at least 161 of the children (83%) had received BCG vaccination. As the Western Cape has also been shown to have the highest incidence of TBM in South Africa, there is concern that BCG as currently used does not have a significant protective effect against disseminated tuberculosis. Seventy-seven children (40%) were also reported to have a close household contact who had been treated for pulmonary tuberculosis within the previous 24 months. Only 17 of these children (22%), however, were prescribed prophylactic isoniazid and only 7 of these completed 3 months or more prophylaxis.

Commercial surrogacy and the commodification of children: an ethical perspective.

The aim of this article is to establish whether there is anything intrinsically immoral about surrogacy arrangements from the perspective of the children born from such arrangements. We focus on the claim that surrogacy entails the wrongful commodification or transfer of children, thereby degrading them and disrespecting their inherent moral value as individual human beings.

Raised intracranial pressure, the syndrome of inappropriate antidiuretic hormone secretion, and arginine vasopressin in tuberculous meningitis.

Intracranial pressure (ICP) monitored shortly after admission over a period of 1 h in 31 children with tuberculous meningitis (TBM) was significantly higher (median 22.5 mmHg, range 8.4-50.9 mmHg) in 19 children with laboratory evidence of the syndrome of inappropriate antidiuretic hormone secretion (SIADH) than in 12 children without such evidence (median 16.2 mmHg, range 5.8-42.5 mmHg; P = 0.027). Neither plasma nor cerebrospinal fluid arginine vasopressin (AVP) was related to ICP (r = 0.33 and 0.13 respectively). Mean arterial pressure (MAP) was measured in 23 children and a moderate correlation was found with plasma AVP (r = 0.62; P = 0.0019). In TBM, plasma AVP may be secreted as a response to raised ICP in an effort to raise MAP and maintain cerebral perfusion pressure. In this setting excess fluid may be inappropriately retained, leading to hyponatremia and hypo-osmolemia.

Plasma arginine vasopressin and the syndrome of inappropriate antidiuretic hormone secretion in tuberculous meningitis.

Biochemical evidence of the syndrome of inappropriate antidiuretic hormone secretion (SIADH) was documented in 17 of 24 (71%) children with tuberculous meningitis. Plasma arginine vasopressin concentrations in patients with signs of SIADH were significantly higher (median, 15.44 (range, 1.62 to greater than 24.52) pg/ml; n = 14) than those without (median, 1.91 (range, 0.44 to 4.91) pg/ml; n = 6) (P less than 0.002). Patients who developed evidence of SIADH were older than those who did not (median, 34 (range, 6 to 101) months vs. 10 (range, 6 to 38) months; P less than 0.007). Five patients with and none without died. In 9 patients evidence of SIADH developed only after hospitalization. These patients received a median of 58 (range, 28 to 109) ml/kg/day fluids (n = 7) before developing evidence of SIADH compared with 107 (range, 58 to 146) ml/kg/day received by patients who did not develop SIADH (n = 6) (P = 0.035). SIADH occurs commonly and its presence appears to influence the outcome of tuberculous meningitis in children.

Tuberculous hydrocephalus: comparison of different treatments with regard to ICP, ventricular size and clinical outcome.

The effect of different treatment regimes on intracranial pressure (ICP), degree of hydrocephalus and clinical outcome was evaluated in 81 children with tuberculous meningitis. 24 children underwent CSF shunting, while 57 with communicating hydrocephalus were randomly assigned to three treatment groups: antituberculous drugs only; or additional intrathecal hyaluronidase or oral acetazolamide and furosemide in addition to antituberculous treatment. The addition of acetazolamide and furosemide was significantly more effective in achieving normal ICP than antituberculous drugs alone. No difference was found in mortality or number of disabled survivors between groups. Of those surviving, nearly two-thirds with stage II tuberculous meningitis were mildly disabled and nearly one-half with stage III were severely disabled at follow-up, emphasising the need for early diagnosis of tuberculous meningitis in the young child.