Germline ATM variants predispose to melanoma: a joint analysis across the GenoMEL and MelaNostrum consortia.

PURPOSE: Ataxia-Telangiectasia Mutated (ATM) has been implicated in the risk of several cancers, but establishing a causal relationship is often challenging. Although ATM single-nucleotide polymorphisms have been linked to melanoma, few functional alleles have been identified. Therefore, ATM impact on melanoma predisposition is unclear. METHODS: From 22 American, Australian, and European sites, we collected 2,104 familial, multiple primary (MPM), and sporadic melanoma cases who underwent ATM genotyping via panel, exome, or genome sequencing, and compared the allele frequency (AF) of selected ATM variants classified as loss-of-function (LOF) and variants of uncertain significance (VUS) between this cohort and the gnomAD non-Finnish European (NFE) data set. RESULTS: LOF variants were more represented in our study cohort than in gnomAD NFE, both in all (AF = 0.005 and 0.002, OR = 2.6, 95% CI = 1.56-4.11, p < 0.01), and familial + MPM cases (AF = 0.0054 and 0.002, OR = 2.97, p < 0.01). Similarly, VUS were enriched in all (AF = 0.046 and 0.033, OR = 1.41, 95% CI = 1.6-5.09, p < 0.01) and familial + MPM cases (AF = 0.053 and 0.033, OR = 1.63, p < 0.01). In a case-control comparison of two centers that provided 1,446 controls, LOF and VUS were enriched in familial + MPM cases (p = 0.027, p = 0.018). CONCLUSION: This study, describing the largest multicenter melanoma cohort investigated for ATM germline variants, supports the role of ATM as a melanoma predisposition gene, with LOF variants suggesting a moderate-risk.

Secondary solid cancer screening following hematopoietic cell transplantation.

Hematopoietic stem cell transplant (HCT) recipients have a substantial risk of developing secondary solid cancers, particularly beyond 5 years after HCT and without reaching a plateau overtime. A working group was established through the Center for International Blood and Marrow Transplant Research and the European Group for Blood and Marrow Transplantation with the goal to facilitate implementation of cancer screening appropriate to HCT recipients. The working group reviewed guidelines and methods for cancer screening applicable to the general population and reviewed the incidence and risk factors for secondary cancers after HCT. A consensus approach was used to establish recommendations for individual secondary cancers. The most common sites include oral cavity, skin, breast and thyroid. Risks of cancers are increased after HCT compared with the general population in skin, thyroid, oral cavity, esophagus, liver, nervous system, bone and connective tissues. Myeloablative TBI, young age at HCT, chronic GVHD and prolonged immunosuppressive treatment beyond 24 months were well-documented risk factors for many types of secondary cancers. All HCT recipients should be advised of the risks of secondary cancers annually and encouraged to undergo recommended screening based on their predisposition. Here we propose guidelines to help clinicians in providing screening and preventive care for secondary cancers among HCT recipients.

Children as hematopoietic cell donors in research: when is it approvable?

With increasing frequency, allogeneic hematopoietic cell transplantation involving children is being performed in the research setting. Allogeneic hematopoietic cell transplantation, however, cannot be performed without a hematopoietic stem cell (HSC) donor. This donor is often a sibling of the recipient and may also be a child. In such circumstances, it is unclear whether or how the federal regulations for pediatric research apply to the minor donors. This introductory paper reviews the issues to be considered while evaluating studies that use HSCs obtained from minor donors and identifies areas where further research is needed. In the era of increasing applicability for donor-derived cellular therapies, we provide a suggested framework for determining when minor donors qualify as human research subjects and when their participation can be approved under the federal regulations.

Hydroxyapatite-ceramic-coated femoral components in young patients followed-up for 16 to 19 years: an update of a previous report.

In 2004 we described the ten-year prospective results of 38 total hip replacements using the Furlong hydroxyapatite-ceramic-coated femoral component in 35 patients < 50 years old. We have now reviewed the surviving 35 arthroplasties in 33 patients at a mean of 16 years (10.3 to 19.9). The mean age of the surviving patients at the time of operation was 41.3 years (26.0 to 49.0). Of these, eight have undergone revision of their acetabular component for aseptic loosening. None of the femoral components has had revision for aseptic loosening giving a survival rate of 100% at 16 years (95% confidence interval 89% to 100%). The Furlong hydroxyapatite-ceramic-coated femoral component gives excellent long-term survival in young and active patients.

Birmingham hip resurfacing arthroplasty: a series of 110 consecutive hips with a minimum five-year clinical and radiological follow-up.

We report the outcome at a minimum of five years of 110 consecutive metal-on-metal Birmingham Hip Resurfacing arthroplasties in 98 patients. The procedures were performed between October 1999 and June 2002 by one surgeon. All patients were followed up clinically and radiologically. The mean follow-up was 71 months (60 to 93). Revision of either component was defined as failure. The mean Harris Hip score at follow-up was 96.4 (53 to 100). The mean Oxford hip score was 41.9 (16 to 57) pre-operatively and 15.4 (12 to 49) post-operatively (p < 0.001). The mean University of California Los Angeles activity score was 3.91 (1 to 10) pre-operatively and 7.5 (4 to 10) post-operatively (p < 0.001). There were four failures giving a survival at five years of 96.3% (95% confidence interval 92.8 to 99.8). When applying a new method to estimate narrowing of the femoral neck we identified a 10% thinning of the femoral neck in 16 hips (14.5%), but the relevance of this finding to the long-term outcome remains unclear. These good medium-term results from an independent centre confirm the original data from Birmingham.

A real-time approach to detect seal defects in food packages using ultrasonic imaging.

The microbial integrity of many types of flexible food packages depends on a zero defect level in the fused seam seal. Human inspection for defects in these seals is marginal at best, and secondary incubation protocols are often used to spot packages with compromised integrity before releasing product for sale. A new type of inspection method has been developed and is being evaluated for robustness. The purpose of the study was to evaluate a new raster scanning geometry to simulate continuous motion, online ultrasonic inspection of the seal region in flexible food package seals. A principal engineering tradeoff of scanning inspection systems is between increased line speed that results from decreased spatial sampling (less acquired data to process) and decreased image quality. The previously developed pulse-echo Backscattered Amplitude Integral (BAI) mode imaging technique is used to form ultrasound images using the new scanning geometry. At an ultrasonic frequency of 22.9 MHz, 38- and 50-microm-diameter air-filled channel defects in all-plastic transparent trilaminate are evaluated. The contrast-to-noise ratio (CNR) of the processed BAI-mode image is used to quantify image quality as a function of spatial sampling. Results show seal defects (38- and 50-microm diameter) are still detectable for undersampled conditions, although image quality degrades as spatial sampling decreases. Further, it is concluded that the raster scanning geometry is feasible for online inspection.

Shape of the acromion: congenital or acquired--a macroscopic, radiographic, and microscopic study of acromion.

Debate continues as to whether the differing shapes of the acromion are congenital or acquired. This has been investigated by neonatal cadaver study, adult cadaver study, radiographic study, magnetic resonance imaging study, or various other means. No one, to our knowledge, has investigated this by histologic study. A macroscopic and histologic study of 22 cadaveric shoulder joints was carried out to establish what, if any, developmental changes occur in the differing patterns of acromion. The cadaveric shoulders were dissected and examined macroscopically. All of the acromion processes were transected and photographed, and the histology of the anterior and inferior surfaces was studied. In all of the curved and hooked types of acromion, a common pattern of degeneration of collagen, fibrocartilage, and bone was observed, consistent with a traction phenomenon. None of the flat acromions exhibited these changes. Extensive histologic changes were noted on the anterior surface of acromion as compared to the inferior surface in curved or hooked acromion. We conclude that the different shapes of acromion are, therefore, acquired as a response to traction forces applied via the coracoacromial ligament and are not congenital in origin.

Sub-biochemical hypothyroidism: an exaggerated thyroid stimulating hormone response to thyrotrophin releasing hormone.

BACKGROUND: The availability of sensitive and specific assays for evaluation of the thyroid axis has allowed definition of thyroid disorders at subclinical stage. This has almost obviated the use of thyrothrophin releasing hormone (TRH) study. We describe here a group of patients with minimal signs of hypothyroidism having normal thyroid function tests (T3, T4, thyroid stimulating hormone (TSH)) and have shown exaggerated TSH response to TRH. MATERIAL AND METHODS: Total 82 subjects were studied. Of these, 11 were age and sex matched controls, and 71 were patients. In all subjects TSH and other thyroid assays (T3, T4, FT4) were done by immunoradiometric assay (IRMA), and radioimmunoassay (RIA) respectively. Thyroid antibody was carried out by haemagglutination method. Results were compared to age and sex related normal ranges. To further investigate the status of thyroid axis, TRH study was carried out using standard protocol. RESULTS: Based on TRH study patients were grouped in three categories. Group 1 included 29 patients whose TSH response to TRH was normal. Group 2 included 20 patients with normal baseline TSH and exaggerated TSH response to TRH and Group 3 included 18 patients with baseline TSH in the range of 5 to 10 mu IU/ml and exaggerated TSH response to TRH. There was a significant difference to total T3 between group 1 and 3 (p < 0.05) but mean values were within normal limits. While no significant difference was observed in total T4 between controls and patient's group. Serum TSH values were high in group 3 as compared to controls and Group 1 and 2 (p < 0.0001). For Free T4 no statistical significance was observed between Group 1, 2 and 3. Thyroid antibodies were positive in 22.7% of patients in Group 2 and 33.33% in Group 3. CONCLUSION: We conclude from the present study that even with sensitive TSH assays TRH study still has a role to mark the early stage of hypothyroidism. Those with a normal or upper normal TSH with exaggerated response to TRH are termed as sub-biochemical hypothyroidism and can be considered for thyroid replacement therapy.

Optimization of a hydrophobic solid-phase extraction interface for matrix-assisted laser desorption/ionization.

Matrix-assisted laser desorption/ionization (MALDI) probe surfaces derivatized with octadecanethiol (C18) can be used as hydrophobic solid-phase extraction devices to isolate and desalt biopolymers directly on the probe surface. Using quantitative MALDI, it was possible to determine the approximate amount of peptide that bound to C18 surfaces and thus to calculate a surface density. It was determined that the amount of peptide bound at the probe surface was independent of the analyte concentration in the immersion solution (from high- to sub-ng ml-1 concentrations), but rather was dependent on the immersion time of the surface as it was exposed to the analyte. The capacity of C18-derivatized probes to bind biopolymers in fixed amounts frees the analyst from the necessity for adjusting analyte concentration through multiple step procedures such as serial dilution or vacuum drying. This time savings result in an overall increase in the efficiency of the MALDI technique.

Differential effects of estradiol and its analogs on cyclin D1 and CDK4 expression in estrogen receptor positive MCF-7 and estrogen receptor-transfected MCF-10AEwt5 cells.

Estradiol stimulates the growth of a majority of human breast tumors containing the estrogen receptors (ERs), proteins that mediates estrogen function. Opposing effects of estradiol have been found in cells expressing endogenous ER and those containing a transfected ER. To understand the role of estradiol structure in diverse estrogenic responses related to cell cycle regulation, we evaluated the effects of estradiol and its analogs on cell cycle progression, and the expression of cyclin D1 and the cyclin dependent kinase 4 (CDK4) in ER-positive MCF-7 and ER-transfected MCF-10AEwt5 cells. Four analogs of estradiol, with a re-positioned or a deleted hydroxyl group, were used. Our results show that estradiol and all of the analogs facilitated cell cycle progression of MCF-7 cells. In contrast, only estradiol inhibited the cell cycle progression of MCF-10AEwt5 cells significantly. Western blot analysis revealed that cyclin D1 protein increased to the maximal level by 6 h after the initiation of cell cycle from G1 phase of MCF-7 cells. The least effective analog in inducing cyclin D1 was 3-hydroxyestratriene. However, this analog was most effective at inducing CDK4, contributing to its efficacy in facilitating MCF-7 cell cycle. In contrast to MCF-7 cells, the level of cyclin D1 protein was not influenced significantly by estradiol or its analogs in MCF-10AEwt5 cells. Sucrose gradient sedimentation analysis of ER from MCF-7 cells showed that the major peak of [3H]-estradiol bound to ER could be displaced by a 10-fold excess of unlabelled estradiol or any of the analogs. In contrast, several analogs were less effective than unlabelled estradiol in competitive displacement of [3H]-estradiol bound to ER from MCF-10AEwt5 cells. These data indicate that the induction of cyclin D1 is an important part of the growth stimulatory effects of estrogens in MCF-7 cells, but it may not be involved in growth inhibition of MCF-10AEwt5 cells. Our results also show that estrogenic compounds interact with ER from MCF-10AEwt5 cells with altered ligand binding affinity, possibly due to the absence or dysfunction of certain transcription factors or ER-associated proteins that co-regulate ER function.

Alprazolam-reinforced medication use in outpatients with anxiety.

The reinforcing effects of alprazolam were investigated in 14 patients who had generalized anxiety or panic disorder, but were not current users/abusers of other psychoactive substances. Using a double-blind outpatient choice procedure, color-coded alprazolam (0.5 mg) and placebo capsules were provided to patients for use 'as needed' in the treatment of anxiety symptoms. Comparisons of alprazolam and placebo during a 2 week sampling period in which placebo and alprazolam were available sequentially revealed no significant differences on measures of medication usage or anxiety levels, although alprazolam did increase subjective ratings of drug effects side effects. During a 4 week choice period, alprazolam was strongly preferred over placebo in 11 out of 14 patients indicating that alprazolam functioned as a reinforcer. Medication usage ranged from zero to 4.0 mg alprazolam in a day. Variations in daily medication-use were positively correlated with anxiety level fluctuations for a majority of patients. For a majority of patients, the results indicate that alprazolam functioned as a reinforcer without accompanying signs of abuse or addiction.

Structural brain scanning in psychiatric patients: a further look.

BACKGROUND: Indications for computed tomographic (CT) and magnetic resonance (MR) scans of the brain in psychiatric patients are not clearly defined. This survey attempted to refine indications suggested by an earlier survey. METHOD: All brain scans ordered in a psychiatric teaching hospital were surveyed over a period of approximately 23 months. A revised order sheet provided information about the assumed need for scanning as well as indicated those factors from the previous survey most likely to reveal pertinent information. Scan results were later correlated with this information. RESULTS: Of 68 scans reviewed, 40 were normal, 16 equivocal, and 12 were abnormal. Cognitive decline and/or confusion were indications for 30 scans; 7 were abnormal, all meriting a clinical diagnosis of Alzheimer dementia. Only 1 of 12 scans to investigate a history of head injury was abnormal. Only 2 of 11 scans based on a history of seizures were abnormal; all had other possible indications. In one patient a prior stroke was confirmed, and in another, active multiple sclerosis. Ten of the group of 12 abnormal scans were found in patients over the age of 40 years, emphasizing the influence of age. CONCLUSION: Revised indications based on this second survey were as follow: (1) The importance of new or unexplained focal neurologic signs was confirmed; (2) Scans are useful for bolstering a clinical diagnosis of Alzheimer disease; (3) A first psychotic break or personality change after the age of 50 years will require investigation; (4) Cognitive decline in a patient with a prior history of functional psychosis is most likely part of the psychosis rather than a confounding organic disorder; (5) Isolated seizures of long duration or abnormal EEG without other clinical findings are seldom associated with abnormal scans.

Batch cultivation of Methylosinus trichosporium OB3b: V. Characterization of poly-beta-hydroxybutyrate production under methane-dependent growth conditions.

Methanotrophs have promising applications in the bioremediation of chlorinated hydrocarbons and in the production of a biopolymer, poly-beta-hydroxybutyrate (PHB). Batch bioreactor culture conditions were studied for the accumulation of PHB by methane-grown Methylosinus trichosporium OB3b, and to evaluate the effect of PHB on the bacterial capacity to degrade trichloroethylene (TCE), a common groundwater contaminant. The PHB content of the washed and lyophilized cells was measured by gas chromatography (GC), after hydrochloric acid (HCl) propanolysis. A differential GC-based assay was developed for the monomer and the polymer of beta-hydroxybutyrate utilizing 1% and 10% HCl (v/v) reaction mixtures, respectively. During bioreactor growth in a Cu-deficient modified Higgins' medium, the cells accumulated PHB upon depletion of nitrate. A biomass yield of 3.2 g dry wt/L and a PHB accumulation of approximately 10% (w/w) were reached after 140 to 160 h, without adversely affecting the propene or TCE epoxidation specific rate given by whole cells containing soluble methane monooxygenase (sMMO). The TCE biotransformation capacity ( approximately 0.25 mg TCE oxidized/mg dry cell wt) of resting cells containing approximately 10% PHB was consistently approximately 1.6-fold greater than that of cells containing only approximately 2% PHB. Higher levels (>10%) of accumulated PHB did not enhance this biotransformation capacity further. By replacing the bioreactor inlet air + CO(2) mixture with pure O(2) at approximately 85 h of batch operation, a PHB accumulation of approximately 45% was achieved after 160 h, but the whole-cell sMMO activity was markedly decreased. In contrast, cells grown in a 10 muM Cu-supplemented Higgins' nitrate minimal salts medium (particulate MMO formation) accumulated up to 50% PHB in only 120 h, coupled with a very high biomass yield of 18 g dry cell wt/L. High PHB accumulations above approximately 20% by both the -Cu and the +Cu grown cells resulted in a decreased ratio of the electronic cell count to the absorbance at 660 nm, which is commonly used to monitor bacterial growth. (c) 1996 John Wiley & Sons, Inc.

Batch cultivation of Methylosinus trichosporium OB3B: IV. Production of hydrogen-driven soluble or particulate methane monooxygenase activity.

Batch culture conditions were established for the formation of H(2)-driven whole-cell soluble or particulate methane monooxygenase (sMMO or pMMO) activity in the obligate methanotroph, Methylosinus trichosporum Ob3b, to expand its potential uses in groundwater bioremediation and the production of specific chemicals. Addition of either Ni and H(2) to a nitrate-containing minimal salts growth medium or Ni and Mo to a nitrate-lacking growth medium (induces a nitrogenase that generates intracellular H(2)) markedly enhanced both the hydrogenase and the accompanying washed-cell H(2)-driven MMO activities of shake-flask cultured cells. For sMMO containing cells, H(2) provided in vitro reducing power for the oxidation of chlorinated solvents such as chloroform and trichloroethylene. Cell cultivations under N(2)-fixing conditions in a 5-L bioreactor, however, required an initial nitrate concentration of at least 1 to 2 mM to achieve high biomass yields (5 to 7 g of dry cell wt/L) for cells producing H(2)-driven sMMO or pMMO activity. Elevation of the initial medium nitrate concentration to 20 mM shortened the culture time for pMMO producing cells by 40%, yet still generated an equivalent growth yield. High nitrate also shortened the culture time for sMMO containing cells by approximately 25%, but it lowered their biomass yield by 26%. Upon storage for 5 weeks at room temperature, washed resting-state cells retained 90% and 70% of their H(2)-driven sMMO and pMMO activity, respectively. This makes their practical use quite feasible. (c) 1995 John Wiley & Sons, Inc.

Cultivation of Methylosinus trichosporium OB3b: III. production of particulate methane monooxygenase in continuous culture.

Continous culture experiments with the obligatory methanotroph, Methylosinus trichosporium OB3b, were conducted to study the whole-cell methane monooxygenase (MMO) and nitrogenase activities in a nitrate minimal salts medium under oxygen-limited conditions with methane as the carbone source. The important variables investigated were the feed medium concentrations of copper and nitrate, CO(2) addition, the agitation speed, and the dilution rate. M. trichosporium OB3b required quantitative amounts of copper (2.6 x 10(-4) g Cu/g dry cell Wt) for the exclusive production of particulate MMo during continuous culture growth. When the feed medium nitrate concentration was varied in the range of 5-50 mM, the whole-cell specific pMMO activity exhibited a maximum at 40 mM. The elimination of external CO(2) gassing decreased pMMO activity by more than 30%. The steady-state cell density increased continuously over a 300-700 rpm range of agitation speed, whereas, the pMMO activity became maximal at 400 rpm. Also, the pMMO activity increased with the dilution rate up to 0.06 h(-1) and remained constant thereafter. Maximal continuous pMMO productivity was, thus, achieved in Higgin's medium containing 10 microM Cu, 80 microM Fe, and 40 mM nitrate with an agitation speed of 500 rpm and a dilution rate of 0.06 h(-1). Nitrogenase activity, on the other hand, increased over a feed medium copper concentration of 2-15 microM, falling sharply at 20 microM, and it exhibited a minimum at 20 mM when the feed medium nitrate concentration was varied.

Batch cultivation of Methylosinus trichosporium OB3b: II. Production of particulate methane monooxygenase.

The obligatory methanotroph, Methylosinus trichosporium OB3b, was studied to optimize the batch culture conditions for the formation of particulate methane monooxygenase (pMMO) in a nitrate minimal salts medium. The important medium components investigated were copper, carbon dioxide, and nitrate. The whole-cell specific pMMO activity decreased sharply with increasing copper concentrations in the range of 10-40 microM and remained constant upon further increases of the copper concentration to 120 microM. The cell growth rate (micro), on the other hand, decreased over the entire range (10-120 microM) of copper concentrations tested. When pMMO was produced in a bioreactor with an optimal initial copper concentration of 10 microM, M. trichosporium OB3b exhibited a much faster overall growth rate and a higher whole-cell propene epoxidation activity compared to our earlier study, in which soluble methane monooxygenase (sMMO) was produced with copper-deficient medium. The addition of external carbon dioxide to the bioreactor culture eliminated an initial lag period in the cell growth. When the standard culture medium nitrate concentration (10 mM) was depleted, the pMMO activity, but not the growth rate, decreased rapidly. The whole-cell specific pMMO activity could be maintained by subsequent supplementation of nitrate. A 4-fold higher initial culture medium nitrate concentration of 40 mM, however, resulted in slower cell growth and lower pMMO activity. These observations demonstrate that, in addition to affecting the exclusive production of pMMO, copper also has an important previously unrecognized role in enhancing the growth rate of M. trichosporium OB3b. They also indicate that for the optimal batch production of pMMO with the minimal medium under study, nitrate should be supplied intermittently during the course of cultivation until other culture medium components become growth-limiting.

Comparative bioavailability of slow release diclofenac (Voveran SR) with enteric coated tablet and internationally used Voltaren Retard.

The objective of the study was to compare the enteric coated diclofenac sodium (Voveran), the slow release formulation developed in India (Voveran SR) and the internationally marketed formulation Voltaren Retard. Ten healthy volunteers were administered 100 mg each of the three formulations in a three-way crossover fashion. Blood samples were collected over 24 hours following administration of the drug; plasma levels of unchanged drug were determined by gas chromatography. Pharmacokinetic parameters for the three formulations were compared. The extent of the drug available from the three formulations was the same as the mean AUC values were not significantly different. Cmax and MRT values for the two slow release formulations were comparable but were significantly different from the values obtained with the enteric coated formulation. Tmax values for the two slow release formulations were similar while the enteric coated tablet had faster time to peak. Voveran SR is comparable to Voltaren Retard and has the distinct advantage of a slow release formulation in that its Cmax is much lower and levels are maintained over 12 hours and detectable upto 24 hours. This slow release formulation will offer clinical advantages of better compliance, relief of early morning symptoms and better tolerability over long term usage.

Partial Purification and Characterization of Two Hydrogenases from the Extreme Thermophile Methanococcus jannaschii.

F(420)-nonreactive and F(420)-reactive hydrogenases have been partially purified from Methanococcus jannaschii, an extremely thermophilic methanogen isolated from a submarine hydrothermal vent. The molecular weights of both hydrogenases were determined by native gradient electrophoresis in 5 to 27% polyacrylamide gels. The F(420)-nonreactive hydrogenase produced one major band (475 kilodaltons), whereas the F(420)-reactive hydrogenase produced two major bands (990 and 115 kilodaltons). The F(420)-nonreactive hydrogenase consisted of two subunits (43 and 31 kilodaltons), and the F(420)-reactive hydrogenase contained three subunits (48, 32, and 25 kilodaltons). Each hydrogenase was active at very high temperatures. Methyl viologen-reducing activity of the F(420)-nonreactive hydrogenase was maximal at 80 degrees C but was still detectable at 103 degrees C. The maximum activities of F(420)-reactive hydrogenase for F(420) and methyl viologen were measured at 80 and 90 degrees C, respectively. Low but measureable activity toward methyl viologen was repeatedly observed at 103 degrees C. Moreover, the half-life of the F(420)-nonreactive hydrogenase at 70 degrees C was over 9 h, and that of the F(420)-reactive enzyme was over 3 h.