X-linked mixed deafness (DFN3): cloning and characterization of the critical region allows the identification of novel microdeletions

We have found that the microsatellite marker AFM207zg5 (DXS995)maps to all previously described deletions which are associatedwith X-linked mixed deafness (DFN3) with or without choroideremiaand mental retardation. Employing this marker and pHU16 (DXS26)we have identified two partially overlapping yeast artificialchromosome clones which were used to construct a complete 850kb cosmid contig. Cosmids from this contig have been testedby Southern blot analysis on DNA from 16 unrelated males withX-linked deafness. Two novel microdeletions were detected inpatients which exhibit the characteristic DFN3 phenotype. Bothdeletions are completely contained within one of the known DFN3-deletions,but one of them does not overlap with two previously describeddeletions in patients with contiguous gene syndromes consistingof DFN3, chorolderemia, and mental retardation. Assuming thatonly a single gene is involved, this suggests that the DFN3gene spans a chromosomal region of at least 400 kb.     

Increased poly(ADP-ribose) polymerase activity in lymphoblastoid cell lines from centenarians

 Poly(ADP-ribosyl)ation is a posttranslational modification of nuclear proteins which is catalyzed by poly(ADP-ribose) polymerase and represents an immediate response of eukaryotic cells to oxidative and other types of DNA damage. Previously a strong correlation had been detected between maximal poly(ADP-ribose) polymerase activity in permeabilized mononuclear leukocytes of various mammalian species and species-specific life span. To study a possible relation between longevity and poly(ADP-ribosyl)ation in humans we measured maximal oligonucleotide-stimulated poly(ADP-ribose) polymerase activity in permeabilized, Epstein-Barr virus transformed lymphoblastoid cell lines from a French population of 49 centenarians and 51 controls aged 20–70 years. Maximal enzyme activity was significantly higher in centenarians than in controls [median of controls: 9035 cpm/10⁶ cells (lower quartile: 6156; upper quartile: 11,410); median of centenarians: 10,380 cpm/10⁶ cells (lower quartile: 7994; upper quartile: 12,991); P=0.031 by Mann-Whitney U test]. In a subset of 16 controls and 24 centenarians, cellular poly(ADP-ribose) polymerase content was determined by quantitative western blotting, thus allowing the calculation of specific enzyme activity. The latter was significantly higher in centenarians (P=0.006), the median value for centenarians being about 1.6-fold that of controls. Specific poly(ADP-ribose) polymerase activity was a more powerful parameter for differentiating between centenarians and controls than enzyme activity relative to cell number. In addition, in a genetic association study we analyzed 437 DNA samples (239 centenarians and 198 controls) by PCR amplification of a polymorphic dinucleotide repeat located in the promoter region of the poly(ADP-ribose) polymerase gene in an attempt to detect an association between this polymorphic marker and variability of enzyme activity or human longevity. However, this genetic analysis revealed no significant enrichment of any of the alleles or genotypes identified among centenarians or controls, but its power was limited by the relatively weak hetero-zygosity of this polymorphic marker in our population (51%). Viewed together with previous results on poly(ADP-ribose) polymerase activity in various mammalian species, the present data provide further evidence for the notion that longevity is associated with a high poly(ADP-ribosyl)ation capacity. Received: 5 September 1997 / Accepted: 10 November 1997     

Synergistic interaction of topographic features in the production of bone-like nodules on Ti surfaces by rat osteoblasts

The objective of this study was to study the responses of osteoblast-like cells to rough Titanium (Ti)-coated epoxy surfaces of differing topographic complexity. Four topographies were studied: polished (PO), coarse-blasted (CB), acid-etched (AE) and coarse-blasted+acid-etched (SLA). Rat osteoblasts were cultured on these surfaces and their morphology, thickness as well as the number and size of bone-like nodules measured. To determine cell shape and cell thickness, fluorescein-5-thiosemicarbazide was used to stain the cell components including the cell membrane, the stained cells were optically sectioned using epifluorescent microscopy and the optical sections were computationally reconstructed to obtain three-dimensional images in which cell volume and cell thickness could be determined. Similarly optical sections of bone-like nodules labeled with tetracycline were also reconstructed to determine their size. The different surface topographies were found to alter the thickness and morphology of osteoblasts cultured on these surfaces. Osteoblasts produced significantly more and larger nodules on SLA compared to other surfaces. Nevertheless and perhaps surprisingly, given the evidence in various cell populations that cell shape can affect cell differentiation, cell thickness was not directly correlated with an increase in bone-like nodule formation. Data were analyzed by factorial analysis of variance. In this way the primary effect of each surface treatment ( i.e. blasting and acid etching) could be assessed as well as their interaction. Both the acid etching and blasting processes significantly affected the number and size of bone-like nodules cultured on Ti surfaces. Moreover there were significant interaction effects indicating that surface topographic features can act synergistically to enhance bone formation. This result suggests that a useful approach to the optimization of surfaces for bone production could involve systematic investigation of combinations of processes each of which produces distinct surface topographical features.     

Novel immunofluorescence assay using recombinant nucleocapsid-spike fusion protein as antigen to detect antibodies against severe acute respiratory syndrome coronavirus

Severe acute respiratory syndrome (SARS) is caused by a novel and highly infectious virus named SARS coronavirus (SARS-CoV). Among the serological tests currently available for the detection of SARS-CoV, a whole-virus-based immunofluorescence assay (IFA) was considered one of the most sensitive assays and served as a "gold standard" during the SARS epidemic in Singapore in 2003. However, the need to manipulate live SARS-CoV in the traditional IFA limits its wide application due to the requirement for a biosafety level 3 laboratory and the risk of laboratory infection. Previously, we have identified two immunodominant epitopes, named N195 and Sc, in the two major structural proteins, the N and S proteins, of SARS-CoV (Q. He, K. H. Chong, H. H. Chng, B. Leung, A. E. Ling, T. Wei, S. W. Chan, E. E. Ooi, and J. Kwang, Clin. Diagn. Lab. Immunol., 11:417-422, 2004; L. Lu, I. Manopo, B. P. Leung, H. H. Chng, A. E. Ling, L. L. Chee, E. E. Ooi, S. W. Chan, and J. Kwang, J. Clin. Microbiol. 42:1570-1576, 2004). In the present study, the N195-Sc fusion protein was highly expressed in insect (Sf9) cells infected with a recombinant baculovirus bearing the hybrid gene under the control of a polyhedrin promoter. An IFA based on Sf9 cells producing the fusion protein was standardized with 23 serum samples from patients with SARS, 20 serum samples from patients with autoimmune diseases, and 43 serum samples from healthy blood donors. The detection rates were comparable to those obtained with a commercial SARS-CoV IFA kit (EUROIMMUN, Gross Groenau, Germany) and a conventional IFA performed at the Singapore General Hospital. Our data showed that the newly developed IFA could detect SARS-CoV in 22 of the 23 SARS-CoV-positive serum samples and gave no false-positive results when the sera from patients with autoimmune diseases and healthy individuals were tested. The detection rate was identical to those of the two whole-virus-based IFAs. Thus, the novel N-S fusion antigen-based IFA could be an attractive alternative to present whole-virus-based IFAs for the diagnosis of SARS-CoV infection.     

A screening study of antidepressant treatment rates and mood symptoms in pregnancy

Summary Study design: As part of a large screening study of perinatal depression, pregnant women were screened for demographic, depression and treatment variables in obstetrics clinics. Women taking antidepressant medication prior to conception were included in the sample as the study aimed to document rates of antidepressant medication use, and relationship to depressive symptomatology.Results: Among women who reported using antidepressant medications within 2 years prior to screening (n = 390, or 11% of all women), 22% reported current use of these medications. Women who reported using antidepressant medications (52%) and those who discontinued them (49%) evidenced elevated depressive symptoms during pregnancy.Conclusions: Both women who discontinue and some who continue antidepressants during pregnancy demonstrate depressive symptoms, suggesting sub-optimal management of both groups. Future studies should carefully assess the adequacy of treatments prescribed as well as the monitoring and adherence of recommended treatments. Full symptom remission should be the goal for antenatal and postnatal depression in order to minimize risk to mother and baby.     

Ectopic bone formation associated with mesenchymal stem cells in a resorbable calcium deficient hydroxyapatite carrier

Bone substitute materials can induce bone formation in combination with mesenchymal stem cells (MSC). The aim of the current study was to examine ectopic in vivo bone formation with and without MSC on a new resorbable ceramic, called calcium deficient hydroxyapatite (CDHA). Ceramic blocks characterized by a large surface (48 m2/g) were compared with beta-tricalcium phosphate (beta-TCP), hydroxyapatite (HA) ceramics (both ca. 0.5 m2/g surface) and demineralized bone matrix (DBM). Before implantation in the back of SCID mice carriers were freshly loaded with 2x10(5) expanded human MSC or loaded with cells and kept under osteogenic conditions for two weeks in vitro. Culture conditions were kept free of xenogenic supplements. Deposits of osteoid at the margins of ceramic pores occurred independent of osteogenic pre-induction, contained human cells, and appeared in 416 MSC/CDHA composites compared to 216 MSC/beta-TCP composites. ALP activity was significantly higher in samples with MSC versus empty controls (p<0.001). Furthermore, ALP was significantly (p<0.05) higher for all ceramics when compared to the DBM matrix. Compared to previous studies, overall bone formation appeared to be reduced possibly due to the strict human protocol. Ectopic bone formation in the novel biomaterial CDHA varied considerably with the cell pool and was at least equal to beta-TCP blocks.     

Do changes in mood and concerns about weight relate to smoking relapse in the postpartum period?

Summary The majority of women who quit smoking during pregnancy will resume smoking during the postpartum period. Little is known, however, about the predictors of postpartum relapses to smoking. Changes in mood and increases in concerns about weight are common during the postpartum period, and these factors may affect womens postpartum smoking behavior. In this paper, we present a model of the relationship among mood, weight concerns and postpartum smoking. Data from previous postpartum relapse prevention trials are reviewed and evidence of a connection between changes in mood and weight concerns to postpartum relapse is presented. Directions for future research on the prevention of smoking relapses during the postpartum period, and the roles of mood and weight concerns in smoking relapse are presented.