Dynamic loading, matrix maintenance and cell injection therapy of human intervertebral discs cultured in a bioreactor.

Low back pain originating from intervertebral disc (IVD) degeneration affects the quality of life for millions of people, and it is a major contributor to global healthcare costs. Long-term culture of intact IVDs is necessary to develop ex vivo models of human IVD degeneration and repair, where the relationship between mechanobiology, disc matrix composition and metabolism can be better understood. A bioreactor was developed that facilitates culture of intact human IVDs in a controlled, dynamically loaded environment. Tissue integrity and cell viability was evaluated under 3 different loading conditions: low 0.1-0.3, medium 0.1-0.3 and high 0.1-1.2 MPa. Cell viability was maintained > 80 % throughout the disc at low and medium loads, whereas it dropped to approximately 70 % (NP) and 50 % (AF) under high loads. Although cell viability was affected at high loads, there was no evidence of sGAG loss, changes in newly synthesised collagen type II or chondroadherin fragmentation. Sulphated GAG content remained at a stable level of approximately 50 µg sGAG/mg tissue in all loading protocols. To evaluate the feasibility of tissue repair strategies with cell supplementation, human NP cells were transplanted into discs within a thermoreversible hyaluronan hydrogel. The discs were loaded under medium loads, and the injected cells remained largely localised to the NP region. This study demonstrates the feasibility of culturing human IVDs for 14 days under cyclic dynamic loading conditions. The system allows the determination a safe range-of-loading and presents a platform to evaluate cell therapies and help to elucidate the effect of load following cell-based therapies.

Selective internal radiation therapy for liver malignancies.

BACKGROUND: Selective internal radiation therapy (SIRT) is a non-ablative technique for the treatment of liver primaries and metastases, with the intention of reducing tumour bulk. This study aimed to determine optimal patient selection, and elucidate its role as a downsizing modality. METHODS: Data were collected retrospectively on patients who underwent SIRT between 2011 and 2014. The procedure was performed percutaneously by an expert radiologist. Response was analysed in two categories, based on radiological (CT/MRI according to Response Evaluation Criteria In Solid Tumours (RECIST)) and biological (α-fetoprotein, carcinoembryonic antigen, carbohydrate antigen 19-9, chromogranin A) parameters. RESULTS: Forty-four patients were included. Liver metastases from colorectal cancer (22 patients) and hepatocellular carcinoma (HCC) (9) were the most common pathologies. Radiological response data were collected from 31 patients. A reduction in sum of diameters (SOD) was observed in patients with HCC (median -24.1 (95 per cent c.i. -43.4 to -3.8) per cent) and neuroendocrine tumours (-30.0 (-45.6 to -7.7) per cent), whereas a slight increase in SOD was seen in patients with colorectal cancer (4.9 (-10.6 to 55.3) per cent). Biological response was assessed in 17 patients, with a reduction in 12, a mixed response in two and no improvement in three. Six- and 12-month overall survival rates were 71 and 41 per cent respectively. There was no difference in overall survival between the RECIST response groups (median survival 375, 290 and 214 days for patients with a partial response, stable disease and progressive disease respectively; P = 0.130), or according to primary pathology (P = 0.063). Seven patients underwent liver resection with variable responses after SIRT. CONCLUSION: SIRT may be used to downsize tumours and may be used as a bridge to surgery in patients with tumours deemed borderline for resection.

Incidence of complex regional pain syndrome after foot and ankle surgery.

Complex regional pain syndrome (CRPS) is an uncommon complication of orthopedic surgery, and few investigators have considered the incidence in foot and ankle surgery. In the present retrospective cohort study of 390 patients who had undergone elective foot and/or ankle surgery in our department from January to December 2009, the incidence of postoperative CRPS was calculated and explanatory variables were analyzed. A total of 17 patients (4.36%) were identified as meeting the International Association for the Study of Pain criteria for the diagnosis of CRPS. Of the 17 patients with CRPS, the mean age was 47.2 ± 9.7 years, and 14 (82.35%) were female. All the operations were elective, and 9 (52.94%) involved the forefoot, 3 (17.65%) the hindfoot, 3 (17.65%) the ankle, and 2 (11.76%) the midfoot. Twelve patients (70.59%) had new-onset CRPS after a primary procedure, and 5 (29.41%) had developed CRPS after multiple surgeries. Three patients (17.65%) had documented nerve damage intraoperatively and thus developed new-onset CRPS type 2. Blood test results were available for 14 patients (82.35%) at a minimum of 3 months postoperatively, and none had elevated inflammatory markers. Five of the patients (29.41%) were smokers, and 8 (47.06%) had had a pre-existing diagnosis of anxiety and/or depression. From our findings, we recommend that middle-age females and those with a history of anxiety or depression, who will undergo elective foot surgery, should be counseled regarding the risk of developing CRPS during the consent process. We recommend similar studies be undertaken in other orthopedic units, and we currently are collecting data from other orthopedic departments within Scotland.

A randomized controlled trial of fallopian tube sperm perfusion compared with standard intrauterine insemination for women with non-tubal infertility.

STUDY QUESTION: Does fallopian tube sperm perfusion (FSP) result in better pregnancy and live birth rates than standard intrauterine insemination (SIUI) for couples with non-tubal infertility with or without gonadotrophin or clomiphene stimulation? SUMMARY ANSWER: There was no evidence of an improvement in live birth rates with FSP compared with SIUI. WHAT IS KNOWN ALREADY: Previous randomized controlled trials have suggested improved live birth rates with FSP but these trials were small. A systematic review published in 2004 suggested heterogeneity in results. STUDY DESIGN, SIZE, AND DURATION: This pragmatic, multicentre, randomized controlled trial compared SIUI and FSP in 417 women with non-tubal infertility. PARTICIPANTS/MATERIALS, SETTING, METHODS: The patients were treated at fertility clinics in New Zealand, Australia and the United Arab Emirates. MAIN RESULTS AND THE ROLE OF CHANCE: Four hundred and seventeen women were randomized to SIUI (n = 210) or FSP (n = 207). Data were available for analysis from 198 women in the SIUI group and 198 women in the FSP group. There were 19 women with incomplete data because of cycle cancellation or withdrawals and 2 women who conceived prior to commencing treatment. There were no significant differences in live birth rates between the two groups with 27 (12.9%) in the SIUI group and 21 in the FSP group (10.1%) [Odds Ratio (OR) 1.31 (0.71, 2.39), P = 0.48]. Two ectopic pregnancies were reported in the SIUI group and one was reported in the FSP group. LIMITATIONS, REASONS FOR CAUTION: Different ovulation protocols were used in the different clinics. Approximately 10% of the cycles involved donor sperm and ∼5% of the cycles did not complete the assigned intervention. WIDER IMPLICATIONS OF THE FINDINGS: There was no evidence of an improvement in live birth rates with FSP compared with SIUI. STUDY FUNDING/COMPETING INTEREST(S): The study was funded in part by the A+ trust of the Auckland District Health Board. No commercial funding was received. TRIAL REGISTRATION NUMBER: ANZCTR Number ACTRN12612001303831.

Posttransplant lymphoproliferative disorder presenting as multiple cystic lesions in a renal transplant recipient.

We report a case of a 67-year-old man who experienced allograft dysfunction following a renal transplantation from a donation after cardiac death. The postoperative course was initially complicated by episodes of E. coli urinary sepsis causing pyrexia and a raised creatinine level. Ultrasound scanning 5 weeks posttransplant revealed mild hydronephrosis with several parenchymal cystic areas measuring up to 2 cm with appearances suggestive of fungal balls. Aspirated fluid again grew Escherichia coli, and this was treated with the appropriate antimicrobial therapy. The patient continued to have episodes of culture-negative sepsis; therefore, a computed tomography scan was performed 6 months posttransplant, which revealed multiple lesions in the renal cortex as well as liver and spleen. Subsequent biopsy revealed an Epstein-Barr virus-driven lymphoproliferation consistent with a polymorphic posttransplantation lymphoproliferative disorder (PTLD). This rare case of PTLD presenting as multiple renal, hepatic and splenic lesions emphasizes the need for a high index of clinical suspicion for this condition. Abnormal para-renal allograft masses should be biopsied to allow swift and effective management of a disease that can disseminate and become significantly more challenging to manage.

Bioaugmentation of pilot-scale slow sand filters can achieve compliant levels for the micropollutant metaldehyde in a real water matrix.

Metaldehyde is a polar, mobile, low molecular weight pesticide that is challenging to remove from drinking water with current adsorption-based micropollutant treatment technologies. Alternative strategies to remove this and compounds with similar properties are necessary to ensure an adequate supply of safe and regulation-compliant drinking water. Biological removal of metaldehyde below the 0.1 µg•L-1 regulatory concentration was attained in pilot-scale slow sand filters (SSFs) subject to bioaugmentation with metaldehyde-degrading bacteria. To achieve this, a library of degraders was first screened in bench-scale assays for removal at micropollutant concentrations in progressively more challenging conditions, including a mixed microbial community with multiple carbon sources. The best performing strains, A. calcoaceticus E1 and Sphingobium CMET-H, showed removal rates of 0.0012 µg•h-1•107 cells-1 and 0.019 µg•h-1•107 cells-1 at this scale. These candidates were then used as inocula for bioaugmentation of pilot-scale SSFs. Here, removal of metaldehyde by A. calcoaceticus E1, was insufficient to achieve compliant water regardless testing increasing cell concentrations. Quantification of metaldehyde-degrading genes indicated that aggregation and inadequate distribution of the inoculum in the filters were the likely causes of this outcome. Conversely, bioaugmentation with Sphingobium CMET-H enabled sufficient metaldehyde removal to achieve compliance, with undetectable levels in treated water for at least 14 d (volumetric removal: 0.57 µg•L-1•h-1). Bioaugmentation did not affect the background SSF microbial community, and filter function was maintained throughout the trial. Here it has been shown for the first time that bioaugmentation is an efficient strategy to remove the adsorption-resistant pesticide metaldehyde from a real water matrix in upscaled systems. Swift contaminant removal after inoculum addition and persistent activity are two remarkable attributes of this approach that would allow it to effectively manage peaks in metaldehyde concentrations (due to precipitation or increased application) in incoming raw water by matching them with high enough degrading populations. This study provides an example of how stepwise screening of a diverse collection of degraders can lead to successful bioaugmentation and can be used as a template for other problematic adsorption-resistant compounds in drinking water purification.

Wnt-1-inducing factor-1: a novel G/C box-binding transcription factor regulating the expression of Wnt-1 during neuroectodermal differentiation.

The Wnt-1 proto-oncogene is essential for proper development of the midbrain and is expressed in a spatially and temporally restricted manner during central nervous system development in mice. In vitro, the gene is specifically transcribed during the retinoic acid (RA)-induced neuroectodermal differentiation of the P19 line of embryonal carcinoma cells. The P19 cells differentiate into neurons, astrocytes, and fibroblast-like cells when treated with RA. Treatment of the cells with dimethyl sulfoxide leads to differentiation along mesodermal lineages, including skeletal and cardiac muscle. We have used the P19 cell line to study the Wnt-1 promoter and identify and characterize the transcription factor(s) that regulates the differentiation-specific transcription of Wnt-1 in RA-treated P19 cultures. Transient-transfection assays have revealed that a 230-bp region comprising positions -278 to -47 of the 5' upstream Wnt-1 sequence was sufficient to direct RA-specific transcription. This promoter fragment was shown to contain a binding site for a nuclear factor that was not detected in undifferentiated P19 stem cells or their dimethyl sulfoxide-treated derivatives but was induced in differentiating RA-treated cells. This factor was termed Wnt-1-inducing factor-1 (WiF-1). DNase I footprinting analysis has identified the G/C-rich WiF-1 binding site, and UV cross-linking studies have shown that WiF-1 is a protein with an M(r) of 65,000. WiF-1 binding activity was also detected in postpubertal mouse testis, the only tissue that expresses Wnt-1 in adults. Site-directed mutations that inhibited WiF-1 binding to the Wnt-1 promoter concomitantly abolished the activity of the promoter in RA-treated P19 cells. The active WiF-1 protein was purified by DNA affinity chromatography. Our data suggest that WiF-1 is a novel G/C box-binding transcription factor and support a physiological role for WiF-1 in the developmentally regulated expression of Wnt-1.

Microbial changes linked to the accelerated degradation of the herbicide atrazine in a range of temperate soils.

Accelerated degradation is the increased breakdown of a pesticide upon its repeated application, which has consequences for the environmental fate of pesticides. The herbicide atrazine was repeatedly applied to soils previously untreated with s-triazines for >5 years. A single application of atrazine, at an agriculturally relevant concentration, was sufficient to induce its rapid dissipation. Soils, with a range of physico-chemical properties and agricultural histories, showed similar degradation kinetics, with the half-life of atrazine decreasing from an average of 25 days after the first application to <2 days after the second. A mathematical model was developed to fit the atrazine-degrading kinetics, which incorporated the exponential growth of atrazine-degrading organisms. Despite the similar rates of degradation, the repertoire of atrazine-degrading genes varied between soils. Only a small portion of the bacterial community had the capacity for atrazine degradation. Overall, the microbial community was not significantly affected by atrazine treatment. One soil, characterised by low pH, did not exhibit accelerated degradation, and atrazine-degrading genes were not detected. Neutralisation of this soil restored accelerated degradation and the atrazine-degrading genes became detectable. This illustrates the potential for accelerated degradation to manifest when conditions become favourable. Additionally, the occurrence of accelerated degradation under agriculturally relevant concentrations supports the consideration of the phenomena in environmental risk assessments.

Cytochrome c' from Paracoccus denitrificans: spectroscopic studies consistent with a role for the protein in nitric oxide metabolism.

Cytochrome c' was purified from the denitrifying bacterium Paracoccus denitrificans and the interaction of the protein with nitric oxide was examined spectroscopically. Two distinct types of haem-nitrosyl electronic absorption spectrum were observed, which were dependent upon [NO]. When cytochrome c' was saturated with NO, alpha and beta bands were centred at 562 nm and 530 nm, whereas with sub-saturating concentrations of NO the alpha and beta bands were red-shifted to 578 nm and 542 nm respectively. Further spectroscopic analysis showed that purified cytochrome c', added to suspensions of P. denitrificans, is able to complex with the NO which is formed as a freely diffusible intermediate of denitrification. In the presence of added NO-3 or NO-2, 40-60% of Fe(II)-cytochrome c' forms a 6-coordinate haem-nitrosyl complex. In the absence of nitrogen oxyanions or NO whole denitrifying cells are able to remove the NO from a Fe(II)-cytochrome c'-NO complex. These findings support the hypothesis that the physiological function of this enigmatic cytochrome involves the reversible binding of nitric oxide.

Crystallization and preliminary crystallographic study of cytochrome cd1 nitrite reductase from Thiosphaera pantotropha.

Single crystals of cytochrome cd1 nitrite reductase from Thiosphaera pantotropha have been grown in the presence of ammonium sulphate by vapour diffusion techniques. Crystals are suitable for X-ray analysis and diffract to dmin = 1.5 A. They belong to space group P2(1) with unit cell dimensions of a = 107.8 A, b = 61.9 A, c = 101.2 A, beta = 112.5 degrees. The crystallographic asymmetric unit contains a dimer.

The 25-hydroxyvitamin D 1-alpha-hydroxylase gene maps to the pseudovitamin D-deficiency rickets (PDDR) disease locus.

Pseudovitamin D-deficiency rickets (PDDR) is an autosomal recessive disorder that may be due to impaired activity of 25-hydroxyvitamin D-1alpha-hydroxylase, a renal cytochrome P450 enzyme (P450[1alpha]) of the vitamin D pathway. The disease locus for PDDR has been mapped by linkage analysis to 12q13-q14, but the molecular defect underlying the enzyme dysfunction has remained elusive due to the lack of sequence information for the P450(1alpha) gene (hereafter referred to as 1alpha-OHase). We have used a probe derived from the rat 25-hydroxyvitamin D-24-hydroxylase (CYP24; 24-OHase) sequence to identify and clone the 1alpha-OHase cDNA. The full-length 1alpha-OHase clone of 2.4 kb codes for a protein of predicted Mr 55 kDa. Functional activity of the cloned sequence was assessed using transient transfection, and the production of authentic 1alpha,25-dihydroxyvitamin D3 [1alpha,25(OH)2D3] was confirmed using high performance liquid chromatography fractionation and time-of-flight mass spectrometry. The expression of the gene was analyzed in vitamin D-replete animals; treatment with 1alpha,25(OH)2D3 reduced 1alpha-OHase transcript levels by 70%, while administration of parathyroid hormone led to a 2-fold increase in the expression of the gene, thus confirming the hormonal regulation previously described using biochemical methods. The rat cDNA was used to obtain a human genomic clone. Interestingly, the human 1alpha-OHase gene mapped to 12q13.1-q13.3, providing strong evidence that a mutation in the 1alpha-OHase gene is responsible for the PDDR phenotype. The availability of a cloned sequence for 1alpha-OHase generates novel tools for the study of the molecular etiology of PDDR, and will allow the investigation of other disturbances of vitamin D metabolism.

The purification of ammonia monooxygenase from Paracoccus denitrificans.

The heterotrophic nitrifier Paracoccus denitrificans expresses a membrane-associated ammonia monooxygenase. The active enzyme has been solubilized in the detergent dodecyl-beta-D-maltoside and purified by standard chromatographic techniques. This is the first purification of an ammonia monooxygenase. The enzyme consists of two subunits with molecular masses of 38 and 46 kDa. The purified enzyme is a quinol oxidase, is inhibited by light and a variety of chelating agents and is activated by cupric ions. These properties indicate that this enzyme has similarities to a family of enzymes including the ammonia monooxygenase from Nitrosomonas europaea and the particulate methane monooxygenase from Methylococcus capsulatus (Bath).

The epidemiology of malaria in a population surrounding Madang, Papua New Guinea.

Malaria is prevalent throughout coastal and lowland Papua New Guinea. Recent changes, including a shift from predominance of Plasmodium vivax to Plasmodium falciparum, appearance of chloroquine-resistant P. falciparum and decreased effectiveness of vector control programs have been observed. Epidemiological features of malaria were studied through four six-month surveys of a population of 16,500 in Madang Province from 1981-1983. Baseline data on parasitology, splenic enlargement, serology, hemoglobin levels, prevalence of 4-aminoquinolines, utilization of mosquito nets and incidence of fever were collected for use in future evaluation of malaria control measures including possible field trials of an antimalarial vaccine. Prevalence of parasitemia (all species, all ages) varied from 35.0% to 42.7% over the four surveys each of which covered a random sample of 25% of the population. The ratio of parasite species was: P. falciparum 70:P. vivax 25:P. malariae 5 in the dry seasons, shifting slightly in favor of P. falciparum during the wet seasons. Intense year-round transmission was indicated by decreasing parasite prevalence and splenic enlargement with age, low density asymptomatic parasitemias and high prevalence of antimalarial antibodies (i.e., greater than 80% of the population over five years of age was ELISA-positive). Levels of endemicity varied geographically, presence of 4-aminoquinolines in urine samples was relatively common (12.7% positive) and chloroquine resistance was widespread (81.6% in vitro, 46.6% in vivo).

Spontaneous mutation of Thiosphaera pantotropha enabling growth on methanol correlates with synthesis of a 26 kDa c-type cytochrome.

Thiosphaera pantotropha grows on methanol as carbon and energy source following spontaneous mutation to a Mox+ phenotype after incubation in media containing methanol. Acquisition of ability to grow on methanol correlates with the appearance of a c-type cytochrome, molecular mass 26 kDa, which is suggested to substitute for the product of the moxG gene, which is the electron acceptor from methanol in related bacteria, but which is absent from T. pantotropha. Mutation leading to growth on methylamine as carbon and energy source was not observed despite the presence of in vitro methylamine dehydrogenase activity in cells grown on choline. Lack of growth on methylamine may correlate with the absence of amicyanin, which is the obligatory electron acceptor from methylamine dehydrogenase in other organisms.

Enantioselective deprotonation reactions using polymer-supported chiral magnesium amide bases.

Novel and readily accessible polymer-supported chiral magnesium amide reagents have been prepared and shown to be effective in the asymmetric deprotonation of a series of prochiral cyclohexanones, affording good to excellent levels of both conversion and enantiomeric ratio (up to 93:7); the Merrifield-based chiral amine species has been shown to be readily recyclable.

Structured role orientation and academic self-concept in the secondary school.

Gordon's (1978) School Environment Preference Survey (SEPS) and Cohen's (1976) six-item adaptation of the Brookover (1967) Self-Concept of Academic Ability Scale were administered to 25 pupils in each of the first four years in a Scottish secondary school. The correlations obtained provide support for Gordon's (1971) hypothesis that pupils with a high degree of structured role orientation, as measured by the SEPS, lack confidence in their academic ability. In addition, there was a decrease in SEPS scores and an increase in academic self-concept over successive year levels.

Thrombocytopenia absent radius syndrome and knee deformity.

We report a case of thrombocytopenia absent radius (TAR) syndrome with varus and flexion deformity and limited movement of the knee. Soft tissue release achieved only partial correction of the deformity and tended to convert a stiff knee to an unstable knee. Flexion deformity required correction by lower femoral extension osteotomy.

Functional bracing of Colles' fractures.

Colles' fracture is one of the most common injuries encountered in orthopaedic practice and yet there is no optimum method for its management. Various operative treatments have been proposed because of the instability of the fracture following reduction. However, considering the epidemiology of the fracture, the majority of these injuries will continue to be treated conservatively with closed reduction and immobilization of the wrist in a cast. Functional bracing is an alternative to cast immobilization, allowing early return of function while maintaining fracture reduction, and is an accepted method of treatment for a number of injuries. Although various methods of functional treatment of Colles' fractures have been described, it has not proved a popular management modality in this injury. We review previous studies of functional bracing and describe a novel method of bracing that we have developed.

The role of voluntary village aides in the control of malaria by presumptive treatment of fever. 1. Selection, training and practice.

As part of a multi-disciplinary malaria research programme in a rural area of Madang Province, the Papua New Guinea Institute of Medical Research (PNG IMR) in 1982 established a village-based intervention programme of presumptive treatment of fever in 35 villages (population about 5,200). Seventy-four villages aides, selected by people from their own village, attended two-week training courses conducted by PNG IMR staff, and were trained to dispense three-day courses of amodiaquine (for children) or chloroquine (for adults) to anyone presenting with fever (presumptive malaria). The majority of village aides, who were voluntary workers, were married men and women between the ages of 20 and 35 years, who had had up to six years' schooling. In 1983, 5,075 fever cases were treated by village aides, which represents a quarter of the number of fever episodes estimated to occur each year in this area. Utilization of village-aide services was variable, the most important determining factors being the personality and standing of the village aide, and the distance (walking time) to the village aide's house from the patient's house. The village aides' role was expanded to include taking blood slides, dispensing other medicines (aspirin and dressings), treatment of diarrhoea by oral rehydration, and registration of vital demographic events in the village. Regular supervision, currently undertaken on a two-weekly basis by PNG IMR staff, regular refresher courses, and, probably, some sort of compensation (not necessarily monetary) are important for the long-term continuation of the programme, which may serve as a model for other areas.(ABSTRACT TRUNCATED AT 250 WORDS)

The role of voluntary village aides in the control of malaria by presumptive treatment of fever. 2. Impact on village health.

A village-based programme of presumptive treatment of fever, using voluntary village aides to dispense oral chloroquine or amodiaquine, was established in 1982 by the Papua New Guinea Institute of Medical Research (PNG IMR) in 35 rural villages or hamlets near Madang, on the north coast of PNG. In the course of the following two years, village aides became an established health resource in many of those villages, although in others they were poorly utilized. In attempting to evaluate the impact of the programme on village health, a number of parameters were investigated. These included malaria-related mortality and morbidity, and the prevalence of parasitaemia and splenic enlargement in children in the study area. Deaths attributable to malaria, which accounted for 11% of deaths in the under-10 year age-group, and cerebral malaria cases were too few to be useful as parameters to evaluate the programme. No reduction in spleen or parasite rates occurred in children as a result of the village aide programme. In two villages, there was an unexplained increase in spleen rate following the introduction of a village aide. A study of malaria-related morbidity, by investigation of all fever cases occurring in a two-week recall period, was conducted in mid-1984. House-to-house interviews were carried out in 19 villages: 9 control villages, where there was no village aide, 6 where the village aide was well utilized, and 4 where the village aide was poorly utilized. The study showed that village aides had a measurable impact on morbidity due to fever in villages where they were well utilized, primarily by reducing the duration of fever through early treatment. The results also suggested that children benefitted even in the villages where the overall utilization of village aides was low. It is felt that such a programme would have had an even greater impact in areas where access to existing health services is more difficult than in the study area.