A recent report suggests that these proteins can also function as

A recent report suggests that these proteins can also function as RNPases [12], which are enzymes that disrupt RNA-protein interactions. Giardia lamblia is a single-celled eukaryotic microorganism check details that inhabits in the upper small intestine of humans and several other vertebrates. Phylogenetic studies have placed Giardia as one of the most early-branching eukaryotic cells [13–17]. In addition to its biological relevance,

G. lamblia is one of the leading causes of human intestinal disease worldwide, the most frequent cause of defined waterborne outbreaks of diarrhea in developed countries and a common cause of diarrhea in daycare centers, institutionalized individuals, backpackers, and travelers [18]. The parasite has a simple life cycle, comprising the disease-causing trophozoites and the environmentally-resistant cysts, which are responsible for the transmission of the disease among susceptible

hosts [18]. Giardia undergoes important adaptive mechanisms to survive both inside and outside the host’s intestine, such as “antigenic variation” and “encystation”, respectively [19]. Antigenic variation is characterized by the continuous switching of surface antigenic molecules, which allows the parasite to evade the immune response generated Gemcitabine datasheet by the host [20]. In Giardia, antigenic variation involves variant-specific surface proteins (VSPs), cysteine-rich type 1a membrane proteins that cover the entire surface of the trophozoites

[21]. Only one VSP, of approximately 200 VSP genes present in the parasite’s genome, is expressed on the surface of individual trophozoites at a given time, but switching to a different VSP occurs once every 6-18 generations. Antigenic variation in Dapagliflozin Giardia is regulated post-transcriptionally by a mechanism similar to RNA interference (RNAi) [22]. Notably, disruption of the RNAi pathway by knocking-down the expression of the dsRNA endonuclease Dicer promotes a change from single to multiple VSP expression on the surface of individual Giardia cells, indicating the direct involvement of this enzyme in controlling antigenic variation in this parasite [23]. Nonetheless, gDicer lacks the C-terminal RNA helicase domain, raising question about the function of this domain in Dicer enzymes of higher eukaryotes. G. lamblia possesses functional RNAi machinery [22]. However, this early-branching eukaryote lacks Drosha and Exportin 5 molecules needed to process and export miRNA from the cell nucleus into the cytoplasm as well as other essential components of the RNAi machinery found in higher eukaryotes [24]. It was recently proposed, however, that lack of Drosha and Exportin 5 in Giardia could be bypassed by the use of snoRNAs as miRNAs precursors [25].

Heart 89:1422–1429CrossRef Roger VL, Weston SA, Redfield MM, Hell

Heart 89:1422–1429CrossRef Roger VL, Weston SA, Redfield MM, Hellermann-Homan JP, Killian J, Yawn BP, Jacobsen SJ (2004) Trends in heart failure incidence and survival in a community-based population. JAMA 292:344–350CrossRef Scottish Intercollegiate Guidelines Network, SIGN 50 (2008) A guideline DAPT molecular weight developer’s handbook revised edition. SIGN, Edinburgh Siegrist J (1996a) Soziale Krisen und Gesundheit (Social crises and health).

Hogrefe Verlag für Psychologie, Göttingen Siegrist J (1996b) Adverse health effects of high effort-low reward conditions. J Occup Health Psychol 1:27–41CrossRef Siegrist J, Peter R, Junge A, Cremer P, Seidel D (1990) Low status control, high effort at work and ischemic heart disease: prospective evidence from blue-collar men. Soc Sci Med Erlotinib datasheet 31:1127–1134CrossRef Siegrist J, Strake D, Chandola T, Godin I, Marmot M, Niedhammer I, Peter R (2004) The measurement of effort-reward imbalance at work: European comparisons. Soc Sci Med 58:1483–1499CrossRef Steptoe A, Hamer M, Chida Y (2007) The effects of acute psychological stress on circulating inflammatory factors in humans: a review and meta-analysis. Brain Behav Immun 21:901–912CrossRef Stewart S, MacIntyre K, Capewell S, McMurray JJ (2003) Heart failure and the aging population: an increasing burden in the 21st century? Heart 89:49–53CrossRef Suadicani P, Hein HO, Gyntelberg F (1993) Are social inequalities as associated with the risk

of ischaemic heart disease a result of psychosocial working conditions? Atherosclerosis 101:165–175CrossRef Theorell T, Floderus-Myrhed B (1977) ‘Workload’ and risk of myocardial infarction—a prospective psychosocial analysis. Int J Epidemiol 6:17–21CrossRef Tsutsumi A, Kayaba K, Hirokawa K, Ishikawa S (2006) Psychosocial job characteristics and risk of mortality in a Japanese community-based working population: the Jichi Medical School Cohort Study. Soc Sci Med 63:1276–1288CrossRef Tsutsumi A, Kayaba K, Kario K, Ishikawa S (2009) Prospective study on occupational stress and enough risk of stroke. Arch Intern Med 169:56–61CrossRef

Tu ST, Wang IW, Lin HF, Liao YC, Lin RT, Liu CS, Hank Juo SH (2010) Carotid intima-media thickness and stiffness are independent risk factors for atherosclerotic diseases. J Investig Med 58:786–790 Uchiyama S, Kurasawa T, Sekizawa T, Nakatsuka H (2005) Job strain and risk of cardiovascular events in treated hypertensive Japanese workers: hypertension follow-up group study. J Occup Health 47:102–111CrossRef Utsugi M, Saijo Y, Yoshioka E, Sato T, Horikawa N, Gong Y, Kishi R (2009) Relationship between two alternative occupational stress models and arterial stiffness: a cross-sectional study among Japanese workers. Int Arch Occup Environ Health 82:175–183CrossRef Vahtera J, Kivimäki M, Pentti J, Linna A, Virtanen M, Virtanen P, Ferrie JE (2004) Organisational downsizing, sickness absence, and mortality: 10-town prospective cohort study.

The “seesaw effect” was first reported as a laboratory phenomenon

The “seesaw effect” was first reported as a laboratory phenomenon by Sieradzki and colleagues [16]. The parent isolate, COL, had a methicillin MIC of 800 mg/L see more with a VAN MIC of 1.5 mg/L; after exposing the isolate to in vitro VAN pressure, MIC increased from 1.5 and 100 mg/L, respectively. The first clinical case describing this type of effect was published 2 years later in a 79-year-old hemodialysis patient with MRSA bacteremia [13]. Initial isolates obtained demonstrated an oxacillin MIC of 3 mg/L and

a VAN MIC of 2 mg/L. After continued VAN exposure and documented sub-therapeutic VAN serum concentrations, the VAN MIC increased to 8 mg/L whereas the oxacillin MIC subsequently decreased to 0.8 mg/L. Similarly, a second case report was published describing a similar effect in a patient with MRSA-infective endocarditis [14]. This patient received a prolonged course of VAN therapy, and as therapy continued the VAN MIC increased from 1 to 8 mg/L while the oxacillin MIC decreased from

as high as 100 to 0.75 mg/L. Additional research on this phenomenon has been carried out utilizing pharmacokinetic/pharmacodynamics in vitro modeling. Werth and colleagues [15] performed in vitro studies evaluating three isogenic S. aureus strain pairs, including DNS and VISA strains exposed to human-simulated concentrations of CPT and VAN. In all three pairs, CPT activity was significantly more active against MRSA strains with reduced glycopeptide susceptibility despite the mutant strains having the same CPT MIC as the parent strains. Though there are in vitro and in vivo data CH5424802 to support the “seesaw effect”, this is the first study to evaluate such a large number of strains including a significant number that are unrelated (all strains except the 8 isogenic strains). The sample of 150 isolates demonstrated a seesaw pattern. These data help to confirm PJ34 HCl the previous observations that have been reported with a few clinical or laboratory-derived strains. As resistance has emerged to antibiotics such as VAN and DAP, the seesaw effect may provide an avenue for alternative

therapeutic options. The seesaw effect can also be further exploited through combination therapy of a glyco- or lipopeptide plus an anti-staphylococcal beta-lactam. In the presence of an anti-staphylococcal β-lactam, DAP binding is increased leading to enhanced depolarization despite increases in DAP MIC [11, 20]. Limitations Potential limitations for this investigation include the evaluation of a limited number of strains and antibiotic combinations utilized in the time–kill curve assessments. Additionally, time–kill curve methodology only utilizes fixed concentration exposures. To further elicit additional impact, multiple dose pharmacokinetic modeling would need to be analyzed. Conclusion In 150 isolates, it was evident that CPT MICs decreased as VAN, TEI, and DAP MICs increased.

nomius and A flavus the most abundant A specific PCR-based meth

nomius and A. flavus the most abundant. A specific PCR-based method for identification at the genus level was developed, which also enabled collective differentiation of the observed section Flavi species

A. flavus, A. nomius and A. tamarii from other Aspergillus species, on the basis of RFLP polymorphism. Given the widespread distribution of Aspergillus section Flavi species and associated risk of food contamination https://www.selleckchem.com/GSK-3.html due to mycotoxin accumulation, simple molecular methods to aid identification of mycotoxigenic species are of importance in identification of CCPs at the point of production and storage, from which appropriate management practices can be developed. Methods Fungal isolation Strains belonging to the genus Aspergillus were isolated from 3 L samples of Brazil nut collected from cooperatives in growing areas in eastern and western regions of the Brazilian Amazon (Amapá, Amazonas and Acre states). A total of three localities were sampled per state. Isolation into pure culture from shell tissues was performed according to Freire et al. [45]. Single spore cultures were used throughout the study, with all strains preserved

both in 20% glycerol at – 80°C and on silica gel at 4°C. Strains were identified to species level based on macroscopic colony morphology and conidial learn more morphology, extrolite production, and sequence data identities for rDNA ITS, β-tubulin Etofibrate and calmodulin gene regions, as described previously

[7, 32, 46]. A representative isolate for each haplotype of each identified Aspergillus species was preserved as a single spore culture and deposited in the reference mycological culture collection at the Department of Phytopathology, University of Brasilia. Determination of aflatoxins and cyclopiazonic acid Analysis of mycotoxigenic potential of a number of Aspergillus section Flavi strains representative of each state was conducted under permissive conditions according to Schmidt-Heydt et al. [47], following growth at 25°C for 7 days on YES medium (20 g/L yeast extract, 150 g/L sucrose, 0,5 g/L MgSO4 5H2O, 0.1 g de ZnSO4, 0.05 g CuSO4,15 g/L agar), with water activity adjusted to 0.99, using a glycerol/water mixture of 108 mL glycerol per litre. Aflatoxin and cyclopiazonic acid standards were acquired from Sigma-Aldrich (Saint Louis, MO, USA), with liquid chromatography grade solvents from Merck (Darmstadt, Germany). For each fungal colony, mycotoxins from the entire content for each colonized plate were extracted under shaking conditions in 10 mL methanol at room temperature for 60 min. Following simple filtration using Whatman No. 1 filter papers, 500 μL of type 1 purified H2O was added to 500 μL of supernatant and filtered through a 0.22 μm teflon membrane. A total of 10 μL of filtrate were diluted with 990 μL of acetonitrile:water (20:80, v/v). The filtrate (10 μL) was then subjected to UPLC/MS/MS analysis.

For MTT assay, MGC-803 cells were seeded in a 96-well plate (Corn

For MTT assay, MGC-803 cells were seeded in a 96-well plate (Corning Costar, Corning, NY, USA) with a density of 5 × 103 cells/well with 10% fetal bovine serum and then cultured overnight. After culturing, those cells were incubated with C-dots CFTR activator of various concentrations for 24 h. Following the incubation, the supernatant was removed and the cells were washed once with 0.01 M PBS. Then 150 μl DMEM and 15 μl MTT stock solution (5 mg/ml in PBS,

pH 7.4) were added to each well, and after this, the cells were allowed to incubate for 4 h at 37°C. Finally, after removing the culture medium, 150 μl DMSO was added to dissolve the Formosan crystals. The optical density (OD) was measured at 570 nm on a standard microplate reader (Scientific Multiskan MK3, Thermo Fisher Scientific, Waltham, MA, USA). The cell viability

was calculated according to the following formula: Cell viability = (OD of the experimental sample/OD of the control group) × 100%. The cell viability of control groups was denoted as 100%. The time-dependent cell response profiles were performed using a real-time cell electronic sensing (RT-CES) system. Firstly, 100 μl of media was added to 16-well E-plates to record background readings, and then, 100 μl of cell suspension (containing about 5,000 cells) was added. Tipifarnib mw Secondly, the cells

in the E-plates were allowed Parvulin to incubate at room temperature for 30 min. After the incubation, the E-plates were put on the reader in the incubator to continuously record the electric impedance which is reflected by cell index. After 20 to 24 h, the RNase A@C-dots and C-dots of certain concentration were added into the E-plates to mix with cells. For comparison, each plate also contained wells added with RNase A and wells with cells alone in the media in addition to media-only wells. The cells were monitored every 2 min for the first 1 h after the addition of C-dots and RNase A to get the short-term response and for every 30 min from 1 h after C-dot addition to about 48 h to record the long-term response. Laser scanning confocal microscopy imaging in vitro For fluorescence imaging with RNase A@C-dots, MGC-803 cells were first plated on 14-mm glass coverslips and allowed to adhere for 24 h at 37°C. Second, the cells were co-incubated with 120 μM RNase A@C-dots for 24 h. Then, the cells were washed with phosphate buffered (PBS) solution to remove unbound nanoparticles. Finally, the cells were fixed with 4% paraformaldehyde, and the nuclei of the cells were stained with 4′,6-diamidino-2-phenylindole (DAPI) (0.5 mg/ml in PBS).

J Trauma 1998, 44:243–252 PubMedCrossRef 10 Gillespie DL, Woodso

J Trauma 1998, 44:243–252.PubMedCrossRef 10. Gillespie DL, Woodson J, Kaufman J, Parker J, Greenfield A, Menzoian JO: Role of arteriography for blunt or penetrating injuries in proximity to major vascular structures: an evolution in management. Ann Vasc Surg 1993, 7:145–149.PubMedCrossRef 11. Ramanathan A, Perera DS, Sheriffdeen AH: Emergency femoral arteriography in lower limb vascular trauma. Ceylon Med J 1995, 40:105–106.PubMed 12. Field CK: Fasciotomy in vascular trauma: Is it too much, too often? Am Surg 1994, 60:409–411.PubMed

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J Vasc Surg 2001, 33:1212–1219.PubMedCrossRef 17. McHenry TP, Holcomb JB, Aoki N, Lindsey RW: Fractures with major vascular injuries from gunshot wounds: implications of surgical sequence. J Trauma 2002, 53:717–221.PubMedCrossRef 18. Wolf YG, Rivkind A: Vascular trauma in high velocity gunshot wounds and shrapnel blast injuries in Isreal. Surg Clin North Am 2002, 82:237–44.PubMedCrossRef 19. Menakuru SR, Behera A, Jindal R, Kaman L, Doley R, Venkatesan R: Extremity vascular trauma in civilian population: a seven-year review from North India. Injury, Int J Care Injured 2005, 36:400–6. 20. Wagner WH, Yellin AE, Weaver FA, Stain SC, Siegel AE: Acute treatment of penetrating popliteal artery trauma: the importance of soft tissue injury. Ann Vasc Surg 1994, 8:557–65.PubMedCrossRef Competing interests The authors declare that they have no competing interests. FER Authors’ contributions RAU CW & WDD performed the listed procedures, collected the data, performed a literature review and drafted the manuscript. SMW analysed the data and critically revised the manuscript. All

authors read and approved the final manuscript.”
“Introduction As soon as surgical access-natural orifice surgery (SA-NOS) has been clearly distinguished from endoscopical access-natural orifice surgery (EA-NOS), being the former more similar to classic laparoscopy and consequently more surgeon-friendly, the trend toward mini-invasiveness has caused a wide dissemination of single port-transumbilical surgical operations [1]. Single port appendectomy (SPA) is gaining quite an interest in the surgical community. Differently from single access cholecystectomy the operation is easily feasible and potentially safe, as the procedure can be carried out approximately in the same manner as the three-port laparoscopic appendectomy (LA)[2].

Tree Physiol 28(1):95–104PubMedCrossRef Dyson-Hudson N (1972) The

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of Khartoum, Sudan Ellis JE, Swift DM (1988) Stability of African pastoral ecosystems—alternate paradigms and implications for development. J Range Manag 41(6):450–459. doi:10.​2307/​3899515 CrossRef El-Sayed R (2004) r’ n Mḏ.iw—lingua blemmyica—tu-bed̨awiε. Ein Sprachenkontinuum im Areal der nubischen Ostwüste und seine (sprach-) historischen Implikationen. Studien zur Altägyptischen Kultur 32:351–362 Fadlalla AH (2007) Embodying Selleck ACP-196 honour. Fertility,

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References 1 Lawson AJ, Logan JM, O’Neill GL, Desai M, Stanley J

References 1. Lawson AJ, Logan JM, O’Neill GL, Desai M, Stanley J: Large-scale survey of Campylobacter species in human gastroenteritis by PCR and PCR-enzyme-linked immunosorbent assay. J Clin Microbiol 1999, 37:3860–3864.PubMed 2. Moore JE, Corcoran D, Dooley JSG, Fanning S, Lucey B, Matsuda M, McDowell DA, Megraud F, Millar BC, O’Mahony R, O’Riordan L, O’Rourke M, Rao RJ, Rooney PJ, Sails A, Whyte P: Campylobacter. Vet Res 36:351–382. 3. Logan JM, Edwards KJ, Saunders NA, Stanley J: Rapid identification

of Campylobacter spp. by melting peak analysis of bioprobes in realtime PCR. J Clin Microbiol 2001, 39:2227–2232.CrossRefPubMed 4. On SL: Identification methods for PFT�� purchase campylobacters, helicobacters and related organisms. Clin Microbiol

Rev 1996, 9:405–422.PubMed 5. Yamazaki-Matsune W, Taguchi M, Seto K, Kawahara R, Kawatsu K, Kumeda Y, Kitazato M, Nukina M, Misawa N, Tsukamoto T: Development of a multiplex PCR assay for identification of Campylobacter coli, Campylobacter fetus, Campylobacter hyointestinalis subsp. hyointestinalis, Campylobacter jejuni, Campylobacter lari and Campylobacter upsaliensis. J Med Microbiol 2007, 56:1467–1473.CrossRefPubMed 6. Debruyne L, On SL, De BrandtE, Vandamme P: Novel Campylobacter lari -like bacteria from humans and molluscs: description of Campylobacter peloridis sp. nov., Campylobacter lari subsp. concheus PF-6463922 ic50 subsp. nov. and Campylobacter lari subsp. lari subsp. nov. Int J Syst Evol Microbiol 2009, 59:1126–1132.CrossRefPubMed 7. Aritomi T, Sekizuka T, Imamaki R, Murayama O, Millar BC, Moore JE, Matsuda M: First restriction and genetic mapping of the genomic DNA of urease-positive thermophilic campylobacters

(UPTC), and small restriction fragment sequencing. Br J Biomed Glutamate dehydrogenase Sci 2006, 63:63–67.PubMed 8. Fouts DE, Mongodin EF, Mandrell RE, Miller WG, Rasko DA, Ravel J, Brinkac LM, DeBoy RT, Parker CT, Daugherty SC, Dodson RJ, Durkin AS, Madupu R, Sullivan SA, Shetty JU, Ayodeji MA, Shvartsbeyn A, Schatz MC, Badger JH, Fraser CM, Nelson KE: Major structural differences and novel potential virulence mechanisms from the genomes of multiple campylobacter species. PLoS Biol 2005, 3:72–85.CrossRef 9. Miller WG, Wang G, Binnewies TT, Parker CT: The complete genome sequence and analysis of the human pathogen Campylobacter lari. Foodborne Pathog Dis 2008, 5:371–386.CrossRefPubMed 10. Burgin AB, Parodos K, Lane DJ, Pace NR: The excision of intervening sequences from Salmonella 23S ribosomal RNA. Cell 1990, 60:405–414.CrossRefPubMed 11. Conlan LH, Stanger MJ, Ichiyanagi K, Belfort M: Localization, mobility and fidelity of retrotransposed group II introns in rRNA genes. Nucleic Acid Res 2005, 33:5262–5270.CrossRefPubMed 12.

During these measurements, the plate was enclosed in a small cham

During these measurements, the plate was enclosed in a small chamber equipped with a window for thermographic measurements to avoid temperature fluctuations and airflow from the incubator. The temperature difference

between a colony and the surrounding medium was determined from the average of the pixels in the infrared image. A typical infrared image is shown in Additional file 1: Figure S3. We also examined the infrared images of colonies grown on a thermal gradient medium. The isolated bacteria stored at −80°C were inoculated in LB broth and incubated at 30°C for 12 hours. After this pre-incubation, 10 μl of the culture medium was inoculated on each 1 cm on LB agar plates (10 × 15 cm) that contained 1% (w/v) glucose. The medium plate was

then click here placed upside down on a table, and a thermal gradient plate (thermal gradient gel electrophoresis system; TITEC Co., Japan) BTK inhibitor was placed on top of the LB agar plate. The temperature of the thermal gradient plate was controlled using two thermocirculator units. After incubation for 2 days under this thermal gradient, infrared images of the LB agar plate were assessed. The surface temperature of the medium was also measured using a thermocouple thermometer (Testo 950, Testo KK) connected to a super-quick action immersion/penetration probe (diameter = 1.5 mm), which had been calibrated using a highly accurate immersion/penetration probe. An infrared image was calibrated using the data from the thermocouple thermometer. Growth rate determinations for strain TK1401 on LB agar Strain TK1401 that had been stored at −80°C was inoculated in LB broth containing 1% (w/v)

glucose and incubated at 30°C overnight. The turbidity of the culture medium was measured at 590 nm and diluted with LB broth containing 1% (w/v) glucose until its optical density at 590 nm was 0.01. Fifty microliters of this culture medium was inoculated onto LB agar plates that contained 1% (w/v) glucose, which were then incubated at 20.0, 22.5, 27.0, 30.0 32.5, and 35.0°C. After incubation, all bacterial cells that grew on ifenprodil the medium plates were harvested as follows. LB broth (1 ml) was poured and bacterial cells on the medium plates were suspended using a spreader. This suspension was collected from the medium plate. Another 1 ml of LB broth was poured on the medium plates and the suspension was collected from the medium plate. Both suspensions were collected and centrifuged at 2,000 × g for 10 min. The bacteria pellet was resuspended in 2 ml of LB broth. The turbidity of the suspension was measured at 590 nm, which was used as an estimate of the number of cells. Determination of the number of bacterial cells that grew on each medium plate was replicated thrice for each incubation time.

A further two centres contributed similar individuals identified

A further two centres contributed similar individuals identified prospectively (Hologic: Guy’s London, Yeovil). Previous case studies of LRP5 HBM used Z-score thresholds to define HBM [13]; however, as Hologic DXA scanner databases store T- but not Z-scores, our search was of T- and/or Z-score ≥ +4. All DXA images were visually inspected by clinicians or clinical

scientists trained in the interpretation of DXA, and those with identifiable explanations for a high BMD value, such as osteoarthritis, were excluded. Evidence of significant www.selleckchem.com/products/cx-4945-silmitasertib.html osteoarthritis on lumbar DXA scans is common. To reduce contamination of our remaining DXA scans by more moderate osteoarthritis, we aimed to refine our case definition based upon restriction to specific lumbar verterba(e).

At our largest centre, 562 scans with T-/Z-score ≥ +4 were graded for OA severity by Kellgren and Lawrence scores and examined in relation to BMD at lumbar vertebral levels [17, 18]. In contrast to other lumbar vertebrae, L1 Z-score was not associated with the presence of OA, reflecting the recognised pattern of progressive OA changes seen in descending sequential lumbar vertebrae [19], nor did total hip Z-score reflect lumbar spine OA. A generalized HBM trait would be expected to affect both spine and hip BMD, though not necessary to the same extent. Hence, we refined our definition of HBM index cases find more as having either (a) L1 Z-score of ≥+3.2 plus a total hip Z-score no lower than +1.2 or (b) a total hip Z-score ≥ +3.2 plus a L1 Z-score no lower than +1.2. A threshold of +3.2 was in keeping with the only published precedent for identifying HBM previously described using DXA [13] and most appropriately differentiated IKBKE generalized HBM from artefact. Z rather than T-score was used to limit age bias. A standard deviation of +3.2 would be expected to identify a tail of 0.069% of a normal distribution [20]. Since the prevalence

of HBM on DXA databases is likely to be influenced by motivations for DXA referral, we examined the latter in a subgroup of 22% of scans at the largest centre in Hull, where referral indication was recorded in an adjunctive database linked to their Lunar DXA database. The distribution of BMD amongst relatives Surviving index cases, identified from DXA database searches described above, who were still resident in the area, were invited by letter and follow-up telephone call to attend their local DXA centre for clinical assessment (described below) and in order to construct family pedigrees. Elderly, immobile individuals were offered home visits to limit participation bias (n = 2).