Acetabular bone defects in THA revision: Reconstruction using morsellised virus-inactivated bone allograft and reinforcement ring. Seven-year outcomes in 95 patients.

BACKGROUND: Acetabular cup loosening is among the main reasons for revision total hip arthroplasty (THA). The implantation of a cryopreserved morsellised bone allograft is a reference method for filling bone defects. However, the outcomes of bone grafts treated with viral inactivation and secured into the host bone (notably using a reinforcement device) are unclear. We therefore retrospectively reviewed cases of acetabular revision with morsellised bone allograft implanted into a reinforcement ring for acetabular revision to assess: (1) clinical survival of the acetabular implant (time to new revision with acetabular component removal), (2) radiological implant survival, (3) and bone graft osseointegration evaluated using Oswestry's criteria. HYPOTHESIS: Virus-inactivated bone allografts provide similar outcomes to cryopreserved allografts. MATERIAL AND METHODS: From 2004 to 2009, 95 patients underwent acetabular revision. There were 60 (63%) females and 35 (37%) males with a mean age of 71.7 years (range: 44.2-90 years). Over 90% of patients had bone defects type 2 or higher in the AAOS classification. Each patient was evaluated after at least 5 years, by an examiner who had not been involved in the revision and who determined the Postel-Merle d'Aubigné (PMA) score and patient satisfaction. We assessed the clinical survival of the acetabular implant (time to new revision with acetabular implant removal), radiological implant survival (migration>5mm, active radiolucent line, failure of graft osseointegration, or reinforcement ring failure), and allograft osteointegration evaluated using Oswestry's criteria. RESULTS: After a mean follow-up of 7years (range: 5.2-10years), 7 (7.4%) patients had been lost to follow-up and 3 (3.4%) had required surgical revision, after 3 to 73 months (for aseptic loosening in 2 cases and infection in 1 case). The estimated 10-year survival rate was 96.2% (95% confidence interval [95%CI]: 88.2-98.7). The mean PMA score at last follow-up had increased significantly, by 2.8 points (p<0.05), to 13.8 (95%CI: 78.4-88.1). Of the 88 re-evaluated patients, 78 (89%) were satisfied or very satisfied. The overall radiological survival rate was 84.5% (95%CI: 78.4-88.1) after a mean of 5.9 years (range: 0.5-10). Allograft osseointegration was satisfactory (Oswestry score≥2) in 95.8% of patients. DISCUSSION: In our population, allografts previously subjected to virus inactivation and implanted into a reinforcement ring produced outcomes similar to those reported previously with cryopreserved allografts. LEVEL OF EVIDENCE: IV, retrospective case-series study.

Tumourigenic non-small-cell lung cancer mesenchymal circulating tumour cells: a clinical case study.

BACKGROUND: Over the past decade, numerous reports describe the generation and increasing utility of non-small-cell lung cancer (NSCLC) patient-derived xenografts (PDX) from tissue biopsies. While PDX have proven useful for genetic profiling and preclinical drug testing, the requirement of a tissue biopsy limits the available patient population, particularly those with advanced oligometastatic disease. Conversely, 'liquid biopsies' such as circulating tumour cells (CTCs) are minimally invasive and easier to obtain. Here, we present a clinical case study of a NSCLC patient with advanced metastatic disease, a never smoker whose primary tumour was EGFR and ALK wild-type. We demonstrate for the first time, tumorigenicity of their CTCs to generate a patient CTC-derived eXplant (CDX). PATIENTS AND METHODS: CTCs were enriched at diagnosis and again 2 months later during disease progression from 10 ml blood from a 48-year-old NSCLC patient and implanted into immunocompromised mice. Resultant tumours were morphologically, immunohistochemically, and genetically compared with the donor patient's diagnostic specimen. Mice were treated with cisplatin and pemetrexed to assess preclinical efficacy of the chemotherapy regimen given to the donor patient. RESULTS: The NSCLC CDX expressed lung lineage markers TTF1 and CK7 and was unresponsive to cisplatin and pemetrexed. Examination of blood samples matched to that used for CDX generation revealed absence of CTCs using the CellSearch EpCAM-dependent platform, whereas size-based CTC enrichment revealed abundant heterogeneous CTCs of which ∼80% were mesenchymal marker vimentin positive. Molecular analysis of the CDX, mesenchymal and epithelial CTCs revealed a common somatic mutation confirming tumour origin and showed CDX RNA and protein profiles consistent with the predominantly mesenchymal phenotype. CONCLUSIONS: This study shows that the absence of NSCLC CTCs detected by CellSearch (EpCAM(+)) does not preclude CDX generation, highlighting epithelial to mesenchymal transition and the functional importance of mesenchymal CTCs in dissemination of this disease.

Patient journey to a specialist amyotrophic lateral sclerosis multidisciplinary clinic: an exploratory study.

BACKGROUND: The multidisciplinary approach in the management of Amyotrophic Lateral Sclerosis (ALS) has been shown to provide superior care to devolved care, with better survival, improved quality of care, and quality of life. Access to expert multidisciplinary management should be a standard for patients with ALS. This analysis explores the patient journey from symptom onset and first engagement with health services, to the initial visit to a specialist ALS Multidisciplinary Clinic (MDC) in Dublin, Ireland. METHODS: A retrospective exploratory multi-method study details the patient journey to the MDC. Data from medical interviews and systematic chart review identifies interactions with the health services and key timelines for thirty five new patients presenting with a diagnosis of ALS during a 6 month period in 2013. RESULTS: The time from first symptom to diagnosis was a mean of 16 months (median 13 months), with a mean interval of 19 months (median 14.6) from first symptoms to arrival at the MDC. The majority of patients were seen by a general practitioner, and subsequently by neurology services. There was an average of four contacts with health services and 4.8 investigations/tests, prior to their first Clinic visit. On the first visit to the MDC patients are linked into an integrated 'system' that can provide specialist care and link with voluntary, palliative and community services as required. CONCLUSIONS: Engagement with a multidisciplinary team has implications for service utilization and quality of life of patients and their families. We have demonstrated that barriers exist that delay referral to specialist services. Comprehensive data recording and collection, using multiple data sources can reconstruct the timelines of the patient journey, which can in turn be used to identify pathways that can expedite early referral to specialist services.

Opening-wedge high tibial osteotomy with a secure bone allograft (Osteopure™) and locked plate fixation: Retrospective clinical and radiological evaluation of 69 knees after 7.5years follow-up.

BACKGROUND: Medial opening-wedge high tibial osteotomy (OWHTO) provides reliable and long-lasting benefits, despite the wide range of wedge-filling and internal-fixation techniques used. The purpose of this work was to assess the clinical and radiological outcomes in a case-series of OWHTO performed using a secure bone allograft and locked plate fixation. HYPOTHESIS: The clinical and radiological outcomes of OWHTO with a high-safety bone allograft and locked plate fixation are similar to those reported in previous case-series studies. MATERIALS AND METHODS: A single-centre retrospective design was used to study 69 knees in 64 patients with a mean age of 51.8years (31-53years) and a preoperative hip-knee-ankle (HKA) angle of 173° (165°-180°). The wedge was filled with secure Osteopure™ bone allograft and fixation was achieved using an Integra Surfix(®) locked plate. Mean follow-up was 7.5years (5-9.3years). Clinical and functional outcomes were assessed by determining the IKS and KOOS-PS scores and recording complications related or unrelated to the allograft. The main criterion for assessing OWHTO survival was the time to revision surgery for symptom recurrence. Radiological assessment criteria were the HKA angle, tibial slope, patellar height, and osteoarthritis grade. GESTO criteria were used to evaluate the behaviour of the allograft. RESULTS: Of the 69 knees, 64 (92.8%) were re-evaluated. The survival rate after 9.3years was 95%±2.7% (3 failures managed with arthroplasty). The functional IKS score improved significantly, by 20 points (P<0.001). Mean increases of 7.8° for the HKA angle and 3.5° for tibial slope were recorded. Bone healing without radiological abnormalities was consistently achieved within 6months. There were no complications related to the allograft (infections, allergies; local or systemic toxicity). DISCUSSION: The clinical, radiological, and safety outcomes documented in our study were similar to those reported in earlier work. LEVEL OF EVIDENCE: IV, retrospective case-series study.

Relative accuracy and availability of an Irish National Database of dispensed medication as a source of medication history information: observational study and retrospective record analysis.

WHAT IS KNOWN AND OBJECTIVE: The medication reconciliation process begins by identifying which medicines a patient used before presentation to hospital. This is time-consuming, labour intensive and may involve interruption of clinicians. We sought to identify the availability and accuracy of data held in a national dispensing database, relative to other sources of medication history information. METHODS: For patients admitted to two acute hospitals in Ireland, a Gold Standard Pre-Admission Medication List (GSPAML) was identified and corroborated with the patient or carer. The GSPAML was compared for accuracy and availability to PAMLs from other sources, including the Health Service Executive Primary Care Reimbursement Scheme (HSE-PCRS) dispensing database. RESULTS: Some 1111 medication were assessed for 97 patients, who were median age 74 years (range 18-92 years), median four co-morbidities (range 1-9), used median 10 medications (range 3-25) and half (52%) were male. The HSE-PCRS PAML was the most accurate source compared to lists provided by the general practitioner, community pharmacist or cited in previous hospital documentation: the list agreed for 74% of the medications the patients actually used, representing complete agreement for all medications in 17% of patients. It was equally contemporaneous to other sources, but was less reliable for male than female patients, those using increasing numbers of medications and those using one or more item that was not reimbursable by the HSE. WHAT IS NEW AND CONCLUSION: The HSE-PCRS database is a relatively accurate, available and contemporaneous source of medication history information and could support acute hospital medication reconciliation.

Exchanges across land-water-scape boundaries in urban systems: strategies for reducing nitrate pollution.

Conservation in urban areas typically focuses on biodiversity and large green spaces. However, opportunities exist throughout urban areas to enhance ecological functions. An important function of urban landscapes is retaining nitrogen thereby reducing nitrate pollution to streams and coastal waters. Control of nonpoint nitrate pollution in urban areas was originally based on the documented importance of riparian zones in agricultural and forested ecosystems. The watershed and boundary frameworks have been used to guide stream research and a riparian conservation strategy to reduce nitrate pollution in urban streams. But is stream restoration and riparian-zone conservation enough? Data from the Baltimore Ecosystem Study and other urban stream research indicate that urban riparian zones do not necessarily prevent nitrate from entering, nor remove nitrate from, streams. Based on this insight, policy makers in Baltimore extended the conservation strategy throughout larger watersheds, attempting to restore functions that no longer took place in riparian boundaries. Two urban revitalization projects are presented as examples aimed at reducing nitrate pollution to stormwater, streams, and the Chesapeake Bay. An adaptive cycle of ecological urban design synthesizes the insights from the watershed and boundary frameworks, from new data, and from the conservation concerns of agencies and local communities. This urban example of conservation based on ameliorating nitrate water pollution extends the initial watershed-boundary approach along three dimensions: 1) from riparian to urban land-water-scapes; 2) from discrete engineering solutions to ecological design approaches; and 3) from structural solutions to inclusion of individual, household, and institutional behavior.

The importance of composite foods for estimates of vegetable and fruit intakes.

OBJECTIVE: To evaluate the contribution of composite foods to vegetable and fruit intakes in Irish adults and to compliance with dietary guidelines for vegetable and fruit intake. DESIGN: Data were analysed from the North/South Ireland Food Consumption Survey of 18-64-year-old adults (n=1379; 662 men, 717 women), which used a 7-day food diary to estimate food intake. RESULTS: The mean intake of vegetables (excluding potatoes) was 140 g day(-1) (men 149 g day(-1); women 132 g day(-1)), of fruit was 136 g day(-1) (men 133 g day(-1); women 140 g day(-1)) and of potatoes was 227 g day(-1) (men 296 g day(-1); women 163 g day(-1)). The mean daily intakes of vegetables, fruit and potatoes from composite foods were 37 g (26%), 6 g (5%) and 17 g (7%), respectively. The mean intake of vegetables from composite foods was unrelated to age or gender, but increased with increasing social class and level of education attained. The proportions of men and women meeting the recommendation for >or=400 g day(-1) (5 servings of 80 g per day) of vegetables and fruit were 21% (15% excluding composite foods) and 19% (12% excluding composite foods), respectively. Compliance with the dietary recommendation decreased with decreasing levels of educational attainment and social class. CONCLUSION: Intakes of vegetables and fruit are low compared with current dietary recommendations, particularly in those of lower levels of educational attainment and social class. Composite foods are an important source of vegetables (less importantly of fruit) and should be included when estimating vegetable intakes. Failure to do so may result in bias in estimates of intake and of compliance with dietary guidelines for population groups, as well as misclassification of individuals by level of intake.

Impact of ready-to-eat breakfast cereal (RTEBC) consumption on adequacy of micronutrient intakes and compliance with dietary recommendations in Irish adults.

OBJECTIVE: To describe the consumption of ready-to-eat-breakfast cereals (RTEBCs) in Irish adults and its impact on adequacy and safety of micronutrient intakes and compliance with dietary recommendations. DESIGN: Analysis for this paper used data from the North/South Ireland Food Consumption Survey that estimated habitual food intake using a 7-day food diary in a representative sample of adults aged 18-64 years ( 662 men, 717 women). RESULTS: Despite the small quantity consumed (mean 28.6 g day-1 or 4.7% of total energy intake), RTEBCs made an important contribution to the mean daily intake of carbohydrate (8.1%), starch (10.8%), dietary fibre (9.8%) and non-starch polysaccharides (NSP) (10.8%) in consumers. Increased consumption was associated with a more fibre-dense diet and with greater compliance with dietary recommendations for fat, carbohydrate and NSP. Fortified RTEBCs contributed significantly to mean daily intakes of iron (18%), thiamin (14%), riboflavin (17%), niacin (15%), vitamin B6 (13%), total folate (18%) and vitamin D (10%) and most of the contribution was from micronutrients added to RTEBCs. Increased consumption of fortified RTEBCs was associated with an increased nutrient density for a number of micronutrients and with a lower prevalence of dietary inadequacy of calcium, iron, riboflavin and folate, particularly in women. However, it was not associated with intakes in excess of the Tolerable Upper Intake Level for any micronutrient. CONCLUSIONS: The consumption of RTEBCs is associated with improved compliance with dietary recommendations for fat, carbohydrate and fibre, with a more micronutrient-dense diet and a reduced risk of dietary inadequacy for calcium, iron, riboflavin and folate, without increasing the risk of excessive intakes of micronutrients.

Evaluation of a spray-dried lacticin 3147 powder for the control of Listeria monocytogenes and Bacillus cereus in a range of food systems.

AIMS: The potential of a powdered preparation of the bacteriocin, lacticin 3147, was investigated for the inhibition of Listeria monocytogenes and Bacillus cereus. METHODS AND RESULTS: A 10% solution of reconstituted demineralized whey powder was fermented with Lactococcus lactis DPC3147 for the generation of a lacticin 3147 containing powdered product. A 99.9% reduction in L. monocytogenes numbers occurred in the presence of the lacticin 3147 powder within 2 h in natural yogurt, and an 85% reduction was observed in cottage cheese within the same time frame. Counts of B. cereus were reduced by 80% in soup, in the presence of 1% (w/w) lacticin 3147 powder, within 3 h. CONCLUSIONS: A powdered preparation of lacticin 3147 was effective for the control of Listeria and Bacillus in natural yogurt, cottage cheese and soup. SIGNIFICANCE AND IMPACT OF THE STUDY: The bioactive lacticin 3147 powder may find broad applications for control of Gram-positive pathogens/spoilage bacteria in a range of foods.

Chronic care clinics for diabetes in primary care: a system-wide randomized trial.

OBJECTIVE: To evaluate the impact of primary care group visits (chronic care clinics) on the process and outcome of care for diabetic patients. RESEARCH DESIGN AND METHODS: We evaluated the intervention in primary care practices randomized to intervention and control groups in a large-staff model health maintenance organization (HMO). Patients included diabetic patients > or = 30 years of age in each participating primary care practice, selected at random from an automated diabetes registry. Primary care practices were randomized within clinics to either a chronic care clinic (intervention) group or a usual care (control) group. The intervention group conducted periodic one-half day chronic care clinics for groups of approximately 8 diabetic patients in their respective doctor's practice. Chronic care clinics consisted of standardized assessments; visits with the primary care physician, nurse, and clinical pharmacist; and a group education/peer support meeting. We collected self-report questionnaires from patients and data from administrative systems. The questionnaires were mailed, and telephoned interviews were conducted for nonrespondents, at baseline and at 12 and 24 months; we queried the process of care received, the satisfaction with care, and the health status of each patient. Serum cholesterol and HbA1c levels and health care use and cost data was collected from HMO administrative systems. RESULTS: In an intention-to-treat analysis at 24 months, the intervention group had received significantly more recommended preventive procedures and helpful patient education. Of five primary health status indicators examined, two (SF-36 general health and bed disability days) were significantly better in the intervention group. Compared with control patients, intervention patients had slightly more primary care visits, but significantly fewer specialty and emergency room visits. Among intervention participants, we found consistently positive associations between the number of chronic care clinics attended and a number of outcomes, including patient satisfaction and HbA1c levels. CONCLUSIONS: Periodic primary care sessions organized to meet the complex needs of diabetic patients imrproved the process of diabetes care and were associated with better outcomes.

The North/South Ireland Food Consumption Survey: the dietary fibre intake of Irish adults.

OBJECTIVE: Mean daily intakes of dietary fibre (DF, Southgate) and non-starch polysaccharide (NSP, Englyst) are estimated in a representative sample of adults aged 18-64 years in Ireland. The contribution of food groups to DF and NSP intake is reported and fibre intakes are compared with dietary recommendations and with intakes in some European countries. DESIGN: Food consumption was estimated using 7-day food diaries for a representative sample (n=1379); 662 men, 717 women) of 18-64 year old adults in the Republic of Ireland and Northern Ireland selected from the electoral register. DF and NSP intakes were estimated from tables of food composition. RESULTS: The mean daily intake of DF in the total sample was 20.2 g (standard deviation (SD) 7.8) [23.2 g (SD 8.5) in men, 17.4 g (SD 5.9) in women] or 2.24 g MJ(-1) (SD 0.7) [2.16 g MJ(-1) (SD 0.7) in men, 2.33 g MJ(-1) (SD 0.7) in women]. Mean daily intake of NSP was 14.8 g (SD 6.2) [16.7 g (SD 6.8) in men, 13.0 g (SD 5.0) in women] or 1.65 g MJ(-1) (SD 0.6) [1.56 g MJ(-1) (SD 0.6) in men, 1.74 g MJ(-1) (SD 0.6) in women]. The main food groups that contributed to mean daily intake of DF (NSP) in the sample were breads 31% (23%), potatoes 19% (23%), and vegetables 17% (19%). Absolute intakes of DF and NSP were higher (P<0.001) in men than women; however, women overall consumed more (P<0.001) fibre-dense diets than men. Women aged 18-35 years consumed less (P<0.01) DF and NSP (g) than women aged 36-64 years. Both men and women aged 18-35 years consumed less (P<0.01) fibre-dense diets than men and women aged 36-64 years. The NSP intake was below the nutritional goal of 18 g day(-1) in 77% of adults and below the minimum of the recommended range (12 g day(-1) in 37% of the total sample. Compliance (i.e. the maximum number of individuals whose collective mean daily intake corresponded to the population goal) with the (UK) population goal for an average intake of 18 g day(-1) NSP was achieved by a greater proportion of the population (63%) than compliance with the (German) dietary fibre recommendation of 30 g day(-1) (27%) or the (Nordic) recommendation of 3 g MJ(-1) day(-1) (33%). CONCLUSION: DF (NSP) intakes were lower than dietary recommendations in a substantial proportion of the population. This is likely to contribute to impaired bowel function and constipation, which in turn may contribute to increased risk of chronic gastrointestinal disease.

Physical activity patterns in a nationally representative sample of adults in Ireland.

OBJECTIVE: To evaluate habitual levels of physical activity in a nationally representative sample of adults in Ireland. DESIGN: Cross-sectional survey using a self-administered questionnaire. Usual levels of work, recreational and household activities were evaluated in relation to anthropometric, demographic and socio-economic characteristics. The amount and intensity of all activities were quantified by assigning metabolic equivalents (METS) to each activity. SETTING: Republic of Ireland and Northern Ireland, 1997-1999. SUBJECTS: Random sample of 1379 adults aged 18-64 years. RESULTS: Men were approximately twice as active in work and recreational activity (139.7 +/- 83.9 METS) as women (68.5 +/- 49.8 METS; P<0.001) but women were three times more active in household tasks (65.9 +/- 58.7 METS vs. 22.6 +/- 24.6 METS; P<0.001). Overall levels of physical activity declined with increasing age, particularly leisure activity in men. In women the decline in work activity was offset by spending more time in household pursuits. Twenty-five per cent of the subjects were extremely overweight (body mass index (BMI>28 kg m(-2)) or obese (BMI >30 kg m (-2)). Fewer obese subjects reported higher levels of work and leisure activities. However, a higher percentage of obese women reported participation in the higher levels of household activities. Participation rates in recreational activities were low. Walking was the most important leisure activity of both men (41%) and women (60%). In terms of hours per week spent in vigorous physical activity, men were more active than women, professional and skilled non-manual women were more active than women in other social classes, and younger subjects (aged 18-35 years) were more active than older subjects. CONCLUSIONS: The holistic approach used in the assessment of physical activity in this study has revealed important and subtle differences in the activity patterns of men and women. Failure to fully characterise the respective activity patterns of men and women could lead to ill-informed public health policy aimed at promoting and sustaining lifetime habits of physical activity. The results suggest that simple population-focused programmes to promote physical activity are unlikely to offer the same chance of long-term success as more sensitive and individualised strategies.

High prevalence of the very rare Wilson disease gene mutation Leu708Pro in the Island of Gran Canaria (Canary Islands, Spain): a genetic and clinical study.

The molecular basis of Wilson disease (WD), an autosomal recessive disorder, is the presence of mutations in the ATP7B gene, a copper transporting ATPase. Hospital records indicated a higher prevalence of WD (1 in 2,600) in some counties in the northeastern region of the island of Gran Canaria (Canary Islands, Spain) that was around 10-fold higher than that described for European populations (1 in 30,000). The ATP7B gene was analyzed for mutations in 24 affected subjects, revealing a high prevalence of the rare Leu708Pro mutation present in 12 homozygous and 7 heterozygous individuals. In these patients, who constitute one of the largest described cohorts of WD homozygotes, we found a variable clinical presentation of the disease, although the biochemical picture was homogenous and characteristic, thereby confirming that the Leu708Pro change is indeed a mutation associated with WD. Haplotype analysis of subjects homozygous for the Leu708Pro mutation showed a conserved shared region smaller than 1 centimorgan (cM), and the region of linkage disequilibrium between the Leu708Pro mutation and neighboring microsatellite markers extended approximately 4.6 cM. When comparing the amount of linkage disequilibrium versus genetic distance from the disease mutation, it was estimated that a common ancestral Leu708Pro chromosome may have been introduced in Gran Canaria over 56 generations ago, dating it back to pre-Hispanic times. The prevalence, and the tight geographical distribution of the Leu708Pro chromosome suggests that the Canary Islands can be considered a genetic isolate for linkage disequilibrium studies.

Evaluation of in vitro effects of 50 and 60 Hz magnetic fields in regional EMF exposure facilities.

A weak association between magnetic-field exposure and increased incidences of cancer has been reported. While alterations in cellular processes after in vitro magnetic-field exposures have also been reported to provide plausibility for this association, other laboratories have been unable to repeat the findings. As part of an accelerated electric- and magnetic-field (EMF) research program, the National Institute of Environmental Health Sciences with the Department of Energy identified the replication of the published positive effects as a priority. Regional EMF exposure facilities were established to investigate major in vitro effects from the literature. These included effects on gene expression, intracellular calcium, colony growth in soft agar, and ornithine decarboxylase activity. The laboratories that first reported these effects provided experimental protocols, cell lines, and other relevant experiment details. Regional facility studies included sham/sham exposures (no applied field in either chamber) and were done in a blinded fashion to minimize investigator bias. In nearly all experiments, no effects of magnetic-field exposure were found. The effort provided insight into dealing with the difficulty of replication of subtle effects in complex biological systems. Experimental techniques provided some clues for the differences in experimental results between the regional facility and the original investigator. Studies of subtle effects require extraordinary efforts to confirm that the effect can be attributed to the applied exposure.

Melanocortin 3 receptor (MC3R) gene variants in extremely obese women.

OBJECTIVE: Following several reports of linkage of obesity related phenotypes to human chromosome 20q we sought to determine whether variations of the melanocortin 3 receptor (MC3R) gene are associated with obesity. DESIGN: We screened the MC3R gene coding region and approximately 2 kb of 5' and 3' flanking sequences for DNA variants in unrelated extremely obese women and average weight controls using polymerase chain reaction (PCR) single strand conformation polymorphism (SSCP) analysis and DNA sequencing. SUBJECTS: 124 unrelated extremely obese women (body mass index, (BMI)>/=40 kg/m2) and 85 average weight controls (BMI<27 kg/m2). MEASUREMENTS: Radiation hybrid (RH) mapping was performed to localize the MC3R gene. 5' and 3' flanking sequences of MC3R gene were cloned. PCR-SSCP and DNA sequencing were used to detect mutations in the MC3R gene coding region and flanking sequences. RESULTS: RH mapping localized the MC3R gene to 20q13, between markers D20S100 and D20S149. 1083 bp 5' and 653 bp 3' flanking region of the MC3R gene were cloned. A missense mutation (+241, codon 81 ATT/GTT, Ile-->Val) was found in the MC3R coding region. Four more variants were detected in the 5' flanking sequence: -201(C-->G), -239 (A-->G), -762(A-->T) and -769(T-->C). Compared with controls, no significant allele frequency differences were found. Racial differences were found for the +241, -201, -239 and -762 polymorphisms. CONCLUSIONS: Several sequence variants were found in the MC3R gene coding region and in 5' flanking sequences. However, none of the variants were associated with obesity phenotypes. The linkage of extreme human obesity on 20q13 is likely caused by genes other than MC3R. International Journal of Obesity (2000) 24, 206-210

Developing applications for lactococcal bacteriocins.

While much of the applied research carried out to date with bacteriocins has concerned nisin, lactococci produce other bacteriocins with economic potential. An example is the two component bacteriocin lacticin 3147, which is active over a wide pH range and has a broad spectrum of activity against gram-positive bacteria. Since the genetic determinants for lacticin 3147 are encoded on a large self-transmissible plasmid, the bacteriocin genes may be conveniently transferred to different lactococcal starters. The resulting food-grade strains can then be used to make a significant impact on the safety and quality of a variety of fermented foods, through the inhibition of undesirable microflora. The bacteriocin is heat stable so it can also be used as an ingredient in a powdered form such as a spray-dried fermentate. Given the observation that lacticin 3147 is effective at physiological pH, there is also considerable potential for biomedical applications. Field trials have demonstrated its efficacy in the prevention of mastitis infections in dairy cows. In contrast to lacticin 3147, the lactococcin bacteriocins A, B and M have a narrow spectrum of activity limited to lactococci. Strains which produce these inhibitors can be exploited in the acceleration of cheese ripening by assisting the premature lysis of starter cultures.

Development of a lacticin 3147-enriched whey powder with inhibitory activity against foodborne pathogens.

The broad-spectrum bacteriocin lacticin 3147, produced by Lactococcus lactis DPC3147, is inhibitory to a wide range of gram-positive food spoilage and pathogenic organisms. A 10% solution of demineralized whey powder was fermented with DPC3147 at a constant pH of 6.5. The fermentate was spray dried, and the resulting powder exhibited inhibitory activity. The ability of the lacticin 3147-enriched powder to inhibit Listeria monocytogenes Scott A and Staphylococcus aureus 10 was assessed in buffer at both acidic (pH 5) and neutral (pH 7) pH. In addition, the ability of the powder to inhibit L. monocytogenes Scott A in an infant milk formulation was assessed. Resuspension of approximately 10(8) midexponential phase L. monocytogenes Scott A cells in a 10% solution of the lacticin 3147-enriched powder resulted in a 1,000-fold reduction in viable cells at pH 5 and pH 7 after 3 h at 30 degrees C. In the case of S. aureus 10, resuspension of 2.5 x 10(7) midexponential phase cells in a 15% solution of the lacticin 3147-enriched powder at pH 5 resulted in only a 10-fold reduction in viable cell counts, compared with a 1,000-fold reduction at pH 7, following incubation for 3 h at 30 degrees C. The use of the lacticin 3147 powder in an infant milk formulation resulted in greater than a 99% kill of L. monocytogenes within 3 h at 30 degrees C. These results suggest that this bioactive lacticin 3147 food ingredient may find applications in many different foods, including those with pH close to neutrality.

Fast and reliable screening of mutations in human tumors: use of multiple fluorescence-based long linker arm nucleotides assay (mf-LLA).

Human tumor samples were screened for point mutations by adapting a mobility-shift assay to automated DNA sizing. This screen identifies the type of point mutation and relative amount of mutated DNA sequences present in a sample. Test samples having known hypoxanthine-guanine phosphoribosyl transferase (hprt)/exon-3 sequence mutations were characterized by: (i) PCR amplification, (ii) fluorescent dye-primer extension with 36-atom linker derived deoxycytosine or deoxyuridine triphosphate and the remaining three natural nucleotides and (iii) sizing of the resulting fluorescently labeled modified strands, using an automated DNA sequencer. Routinely, a range of sizes is observed among the sequence variants of a single DNA target sequence. This is because nucleotide analogs are incorporated into DNA strands in a sequence-dependent manner, resulting in composition-dependent electrophoretic mobility. Thus, point mutations are identified as shifts in mobility between the fluorescently labeled modified strands of the control and test samples. The twenty different hprt/exon-3 single-base substitution mutations tested were easily identified, even at fourfold dilution with control DNA.

Physical mapping of the CXC chemokine locus on human chromosome 4.

A physical map of the CXC chemokine locus on chromosome 4 has been constructed by PCR analysis and PFGE mapping of YAC clones. The genes for IL8, GRO1, PPBP, PF4, SCYB5 (ENA-78) and SCYB6 (GCP-2) have been co-localized on a 335-kb genomic fragment. The GRO2 and GRO3 genes did not map within this region and based on analysis of a YAC contig overlapping IL8 we speculate that GRO2 and GRO3 map downstream of this region. We have also assigned the novel CXC chemokine gene, SCYB9B (alias H174/betaR1) to chromosome 4q21, upstream and within 12 kb of INP10. Like INP10 and MIG, INP10 and SCYB9B are arranged in a head to tail manner. The chromosomal arrangement of these genes appears to reflect the evolution of this multigene family and supports the theory that it arose by gene duplication.