Multiplexed DNA sequencing and diagnostics by hybridization with enriched stable isotope labels

A new DNA diagnostic and sequencing system has been developed that uses time-of-flight resonance ionization mass spectrometry (TOF-RIMS) to provide a rapid method of analyzing stable isotope-labeled oligonucleotides in form 1 sequencing by hybridization (SBH). With form 1, the DNA is immobilized on a nylon membrane and enriched isotope-labeled individual oligonucleotide probes are free to seek out complementary DNAs during hybridization. The major advantage of this new approach is that multiple oligonucleotides can be labeled with different enriched isotopes and can all be simultaneously hybridized to the genosensor matrix. The probes can then be simultaneously detected with TOF-RIMS with high selectivity, sensitivity, and efficiency. By using isotopically enriched tin labels, up to 10 labeled oligonucleotides could be examined in a single hybridization to the DNA matrix. Greater numbers of labels are available if rare earth isotopes are employed. In the present study, matrices containing three different DNAs were prepared and simultaneously hybridized with two different probes under a variety of conditions. The results show that DNAs, immobilized on nylon surfaces, can be specifically hybridized to probes labeled with different enriched in isotopes. Discrimination between complementary and noncomplementary sites of better than 100 was obtained in multiplexed samples. This new SBH method, which employs stable isotopic labels to locate target DNAs and TOF-RIMS to detect the labels, will be a very versatile and extensive multiplexing method.     

Immunologic characterization of CD7-deficient mice

Human CD7 is an Ig superfamily molecule that is expressed on mature T and NK lymphocytes. Although in vitro studies have suggested a role for CD7 in lymphoid development and function, the exact function of CD7 in vivo has remained elusive. One patient has been reported with SCID syndrome attributed to CD7 deficiency. To study in vivo functions of CD7, we have generated CD7-deficient mice and assessed their lymphoid development and function. CD7-deficient mice were viable, had normal peripheral blood and spleen lymphocyte numbers, and had normal specific Ab responses with Ag-driven Ig isotype switching. Thymocyte numbers were normal in 4-wk-old, 6-mo-old, and 1-yr-old CD7-deficient mice, but in 3-mo-old CD7-deficient mice, total thymocyte numbers were significantly increased by 60% (p < 0.02) compared with normal age-matched +/+ littermates. CD7-deficient splenocytes proliferated normally in response to various mitogens, including PHA, anti-CD3, Con A, and LPS. While NK cell numbers and cytolytic activity to YAC targets were normal, CD7-deficient mice had lower Ag-induced MHC class I-restricted CTL activity against OVA-transfected target cells than did +/+ control mice. Thus, CD7-deficient mice did not have a SCID syndrome, but rather had transient increases in thymocyte numbers at age 3 mo and altered splenocyte Ag-specific CTL effecter cell activity. These data suggest a role for CD7 in regulating intrathymic T cell development and in mediating CTL effecter function.     

A wavenumber partitioning scheme for two-dimensional statistical closures

A technique of wavenumber partitioning that conserves both energy and enstrophy is developed for two-dimensional statistical closures. This advance facilitates the computation of energy spectra over seven wavenumber decades, a task that will clearly remain outside the realm of conventional numerical simulations for the foreseeable future. Within the context of the test-field model, the method is used to demonstrate Kraichnan's logarithmically-corrected scaling for the enstrophy inertial range and to make a quantitative assessment of the effect of replacing the physical Laplacian viscosity with an enhanced hyperviscosity.     

Phospholipase A2 activity in salivary glands and saliva of the lone star tick (Acari: Ixodidae) during tick feeding

The etiology of subacute granulomatous thyroiditis (SAT) is obscure, although it is postulated to be associated with viral infections and genetic factors. In the present study, the possibility of an infectious etiology was prospectively studied in 27 consecutive patients with SAT. Special emphasis was put on the role of enteroviruses. Coupled sera (interval one month) were taken from all patients and single sera from 29 control subjects for virus antibody determinations. Stool samples were collected for virus isolation and fine-needle aspiration samples from thyroid gland for the detection of enterovirus RNA using RT-PCR were taken from SAT patients. Enteroviral antibodies were tested using three different methods: indirect EIA, heavy chain capture RIA, and standard complement fixation (CF) test. Antibodies against other common viral pathogens, including enteroviruses, were screened using the CF test and those against Mycoplasma pneumoniae and Chlamydia pneumoniae using EIA and microimmunofluorescence techniques, respectively. Common respiratory viruses were also screened from nasopharyngeal suction samples by antigen detection EIA. Based on serological findings, one patient had acute Cytomegalovirus infection. All other patients were negative in antibody tests, virus isolation, RT-PCR, and antigen detection. Enterovirus RNA was not detected by PCR in the thyroid tissue in any of the fine-needle aspiration samples. There was no evidence of recent enteroviral infections in SAT patients. The results suggest that SAT is not usually associated with acute infections. No evidence was obtained to support the proposed role of enteroviruses as an important etiologic agent of SAT.     

Transcrystalline morphology of nylon 6 on the surface of aramid fibers

In this paper, the isothermal crystallization of nylon 6 in the presence of Kevlar 129 fiber was investigated by polarized optical microscopy (POM). The formation of a transcrystalline domain was found to be mainly controlled by crystallization conditions, such as the temperature of the isothermal crystallization, residual time at melting temperature and the cooling rate of the melt. The nucleation rate of nylon 6 on the fibers was mainly affected by the crystallization temperature. The interfacial transcrystallinity of nylon 6 occurred on the surface of Kevlar 129 fiber in the temperature range 130–190 °C. The reason for the formation of interfacial transcrystalline morphology is discussed from the molecular level, based on the understanding of the packing mode of nylon 6 chains around fibers and the interaction between matrix and fibers. It was found that the lattice matching and hydrogen‐bonding between nylon 6 and poly(p‐phenylene terephthalamide) (PPTA) crystals play an important role in the epitaxial crystallization. Copyright © 2004 Society of Chemical Industry     

Specific, high affinity binding of tissue inhibitor of metalloproteinases-4 (TIMP-4) to the COOH-terminal hemopexin-like domain of human gelatinase A. TIMP-4 binds progelatinase A and the COOH-terminal domain in a similar manner to TIMP-2

The binding properties of the newly described tissue inhibitor of metalloproteinases-4 (TIMP-4) to progelatinase A and to the COOH-terminal hemopexin-like domain (C domain) of the enzyme were examined. We present evidence for the first time of a specific, high affinity interaction between TIMP-4 and the C domain of human gelatinase A and show that TIMP-4 binds both progelatinase A and the C domain in a similar manner to that of TIMP-2. Saturable binding of recombinant C domain to TIMP-4 and to TIMP-2 but not to TIMP-1 was demonstrated using a microwell protein binding assay. The recombinant collagen binding domain of gelatinase A, comprised of the three fibronectin type II-like repeats, did not bind to TIMP-4, indicating that binding is mediated selectively by the C domain. Binding to TIMP-4 was of high affinity with an apparent Kd of 1.7 x 10(-7) M but slightly weaker than that to TIMP-2 (apparent Kd of 0.66 x 10(-7) M). Affinity chromatography confirmed the TIMP-4-C domain interaction and also showed that the complex could not be disrupted by 1 M NaCl or 10% dimethyl sulfoxide, thereby further demonstrating the tight binding. To verify the biological significance of this interaction, binding of full-length progelatinase A to TIMP-4 was investigated. TIMP-4 and TIMP-2 but not TIMP-1 bound specifically to purified TIMP-2-free human recombinant full-length progelatinase A and to full-length rat proenzyme from the conditioned culture medium of ROS 17/2.8 cells. Preincubation of the C domain with TIMP-2 was found to reduce subsequent binding to TIMP-4 in a concentration-dependent manner. Competition between TIMP-2 and TIMP-4 for a common or overlapping binding sites on the gelatinase A C domain may occur; alternatively TIMP-2 may prevent the binding of TIMP-4 by steric hindrance or induction of a conformational change in the C domain. We propose that the binding of progelatinase A to TIMP-4 represents a third TIMP-progelatinase interaction in addition to that of progelatinase A with TIMP-2 and progelatinase B with TIMP-1 described previously. This new phenomenon may be of important physiological significance in modulating the cell surface activation of progelatinase A.     

One-stage hypospadias repair with stent or suprapubic diversion: which is better?

PURPOSE: The authors report on 105 consecutive patients who underwent one-stage hypospadias repair based on use of suprapubic diversion or transurethral drainage with stenting. METHODS: The surgical procedures included 52 metal-based flap urethroplasty (Mathieu) for coronal, subcoronal, and distal shaft hypospadias; 32 transverse island pedicle graft (Duckett) for mid and proximal shaft hypospadias; 21 transverse island pedicle (Duckett) plus rolled midline tube (Thierchs) for penoscrotal and scrotal hypospadias. To accomplish urinary drainage, suprapubic diversion (cystofix) was used in 28 of 52 Mathieu operations, in 17 of 32 Duckett operations, and in 11 of 21 transverse island pedicle graft plus rolled midline tube operations. In the rest of the cases, transurethral drainage with stenting was used. RESULTS: All children had excellent cosmetic and functional outcomes. But the rates of complications such as fistula and meatal stenosis were significantly different between the groups in which suprapubic tube or urethral stent was used. In 56 of the 105 patients in whom suprapubic diversion was used, four (7.14%) had fistulas and three (5.35%) had meatal stenosis, in contrast to a fistula rate of 14.28% and meatal stenosis rate of 12.24% in patients that urethral stent is used for urinary drainage. CONCLUSION: The authors believe that the use of suprapubic diversion is advantageous for the outcome of one-stage hypospadias repair in relation to fistula occurrence and meatal stenosis.     

Two post thinning fertilizer trials inPinus radiata in New South Wales,Australia

Two trials inPinus radiata growing on different sites in N.S.W. allowed consideration of fertilizer applications after 2nd or 3rd thinning. The trials included factorial applications of N and P at a single thinning intensity plus a further treatment which allowed assessment of different thinning intensities. The most significant growth responses were obtained by application of N and P in combination. The largest response (additional productivity compared with the unfertilized control) occurred 4 years after application and after 7 years there was no additional absolute response for either of the two sites. The largest fertilizer response was 70 m³ ha^–1 over 7 years on one site and 36 m³ ha^–1 on the other, indicating differences in absolute responses between sites. It was concluded that in planning treatments the most responsive sites near the end of the rotation should be selected to maximise economic returns. Foliage analyses indicated differences between sites at the commencement of the study. It was concluded that either a single year of foliage analyses at study commencement is of value, or sampling every year of the study should be used to analyse responses, but a single year of analysis during or at the end of the study would not be of value.