Mineral status in cattle fed rice straw and para grass combined with different levels of protein derived from cassava foliage.

Eight male cattle of the Local Yellow breed with an average live weight of 121 kg and an average age of 18 months were used to evaluate the effects of different levels of sun-dried cassava (Manihot esculenta) foliage supplementation on mineral metabolism in growing cattle fed rice straw and para grass as basal diet. Rice straw ad libitum and para grass (Brachiaria mutica) at 1% DM of BW comprised the basal diet. The study was arranged as a 4×4 double Latin square design, with cassava foliage contributing 0, 0.8, 1.6 or 2.4 g CP/kg BW. The cassava foliage intake was lower than the planned levels. DM consumption was significantly affected by cassava foliage supplementation, with the largest intake observed at the two highest levels of cassava foliage supplementation. Rice straw intake showed the opposite pattern, with lower intake at higher cassava foliage supplementation. No refusals occurred for para grass in any of the treatments. Ca, P, Mg, K, S and Mn intake increased significantly with increasing intake of cassava foliage, but Na intake was not affected by treatment. Faecal excretion of Ca, Mg, S and Mn increased significantly with increasing cassava foliage intake. There were no differences between P, K and Na excretion in faeces. There was a significant diet effect on Mg, S and Mn digestibility. Mg and Mn digestibility increased with increasing cassava foliage supplementation, while S digestibility decreased. Ca, P, K and Na digestibility was not affected by diet. There was a significant effect of treatment on P retention, with the highest value observed for supplementation with 1.6 g CP/kg BW cassava foliage. Ca and Mg showed similar trends, with the highest retention again for supplementation with 1.6 g CP/kg BW cassava foliage. There were weak but significant positive correlations between nitrogen retention and the macro minerals Ca, P and Mg. Furthermore, retention of all these minerals was positively correlated. Mineral losses in urine were not affected by dietary treatment with the exception of P excretion, which was affected by treatment. In conclusion, cassava foliage is a good Ca source which compensates for the low Ca content in rice straw and para grass, but P deficiency appears to be exaggerated in cattle with higher cassava intake. The results suggest that under these conditions growing cattle on a high cassava intake would benefit from P and S supplementation.

Feed intake, digestibility, and N retention in cattle fed rice straw and para grass combined with different levels of protein derived from cassava foliage.

Eight male cattle of Local Yellow breed with an average live weight of 121 kg and an average age of 18 months were used to evaluate the effects of different levels of sun-dried cassava foliage supplementation (Manihot esculenta) on intake, digestibility and N retention. Rice straw ad libitum and para grass (Brachiaria mutica) at 1% DM of BW comprised the basal diet. The study was arranged as a 4×4 double Latin square design, with cassava foliage contributing 0, 0.8, 1.6 or 2.4 g CP/kg BW. The cattle selected cassava leaves in preference to petioles. Petiole intake decreased from 64 to 48% of offered petioles when the cassava foliage proportion increased from the lowest to the highest level. The cattle consumed all the leaves at the two lower levels of cassava foliage inclusion and 91% at the highest level. Rice straw intake decreased significantly as the level of cassava foliage increased. Intake of DM, OM, NDF, and ADF increased significantly with increasing intake of cassava foliage. Daily DM intake per 100 kg BW increased from 2.7 to 3.2 kg with increasing cassava foliage intake. No effect on CP digestibility was detected when the level of cassava foliage increased. Digestibility of DM, OM, NDF and ADF was significantly higher in the group fed no cassava foliage than in the other groups. N retention increased from 16 to 28 g/d with the first level of cassava foliage inclusion, but levelled out at the two highest levels. N excretion increased in both faeces and urine as a response to higher intake of cassava foliage. Maximum N retention occurred when 40% of total N intake came from cassava foliage (equivalent to 1.3 g CP/kg BW).

Synaptic targeting of N-methyl-D-aspartate receptor splice variants is regulated differentially by receptor activity.

The formation of postsynaptic clusters of various ligand-gated ion channels is regulated by receptor activity. Here we describe the developmental- and activity-dependent modification of N-methyl-D-aspartate (NMDA) receptor clustering in spinal cord neurons in vitro detected by immunofluorescence analysis using subunit and splice variant specific antibodies. NMDA receptors form synaptic and extrasynaptic clusters with sequential changes in subunit composition during in vitro development. During the first week of in vitro culture, a NR1 splice variant containing the C2-carboxy terminus and lacking the N1-cassette and the NR2B subunit are the prevailing components of receptor clusters at synaptic and extrasynaptic sites. After 3 weeks in culture (days in vitro [DIV] 22), the numbers of postsynaptic receptor clusters with N1-containing NR1 splice variants and NR2A subunits are upregulated. At DIV22, C2-specific clusters are abundant and are predominantly localized at postsynaptic sites, whereas the total number of C2'-clusters in dendrites is much lower and these clusters are localized mostly extrasynaptically. However, upon chronic inhibition of NMDA receptor activity in DIV8 and DIV22 cultures with MK801, the number of postsynaptic NR1-C2' subunit clusters is strongly upregulated. In contrast, numbers of NR1-C2 clusters are only modestly increased in DIV8 and not changed in DIV22 cultures upon MK801 treatment, suggesting a specific role of NR1 carboxy-terminal sequences in the activity-dependent synaptic targeting of NMDA receptor clusters of spinal cord neurons.

Survival of Listeria monocytogenes in wilted and additive-treated grass silage.

Grass was field-dried to 3 different dry matter (DM) levels (200, 430 and 540 g/kg) and inoculated with 10(6)-10(7) cfu/g of a Listeria monocytogenes strain sharing a phagovar occasionally involved in foodborne outbreaks of listeriosis. Formic acid (3 ml/kg) or lactic acid bacteria (8 x 10(5)/g) with cellulolytic enzymes were applied only to forages with low and intermediate DM levels. Forages were ensiled in laboratory silos (1700 ml) and were stored at 25 degrees C for 30 or 90 days. After 90 days of storage, L. monocytogenes could not be detected in any silo, except one with the high dry matter grass without additive. After 30 days of storage, between 10(2) and 10(6) cfu L. monocytogenes/g silage were isolated from the untreated silages. Increasing the DM content from 200 to 540 g/kg did not reduce listeria counts possibly because of the lower production of fermentation acids (higher pH). In silages treated with additives, counts of L. monocytogenes were always lower than in silages without additive. In wet silages (DM 200 g/kg) both additives were effective, but in the wilted silages (DM 430 g/kg) only the bacterial additive reduced listeria counts below detection level. Listeria counts were highly correlated to silage pH (r = 0.92), the concentration of lactic acid (r = -0.80) and the pooled amount of undissociated acids (r = -0.83).

[Synovial membrane diagnostic assessment in rheumatoid arthritis]. / Synovialis-Diagnostik bei Rheumatoider Arthritis.

Rheumatoid arthritis (RA) is the most frequent inflammatory rheumatic disease. At the beginning of the disease, where, based on today's knowledge the therapeutic possibilities are largest, the diagnostic methods do not permit a differentiated estimation of the prognosis. Conventional x-rays are mostly normal and serum markers unspecific. So far--in contrast to other diseases--only little information had been drawn from the pathomorphologic substrate "synovialis" itself to assess the prognosis. Reasons therefor were found in difficulties in obtaining synovial tissue besides surgical interventions, particularly in patients with early arthritis. By minimalizing the diagnostic instruments and improvement of the technique, synovial tissue sampling in RA has become minimally invasive and it is even possible to perform on the smallest joints, such as finger joints. Hereby, synovial analysis is open for detecting pathways of inflammation and joint destruction, which might support the advancement of new therapeutic strategies, followed by a better prognosis and outcome of RA.

Decreased incidence of retinopathy of prematurity, 1995-1997.

PURPOSE: To report a significant decrease in the incidence of retinopathy of prematurity (ROP), both in our neonatal intensive care unit (NICU) and internationally, and review factors in patient care that may be contributory. METHODS: We retrospectively reviewed the records of all neonates weighing less than 1251 g admitted to our NICU from 1995 to 1997 and evaluated the incidence and stage of ROP. These data on 191 neonates were compared with an international NICU database of 9989 similar neonates, which represents all infants who received an ophthalmologic examination in the Vermont-Oxford Network Database (VOND) in 1997, except those from our institute (the University of Kentucky). In addition to investigating the incidence of ROP, we looked at the use of antenatal corticosteroids given 1 to 7 days prepartum, the use of oxygen at 36 weeks' postconceptional age, and the use of oxygen at home upon discharge. RESULTS: In our center, we had a 36.1% incidence of ROP compared with an international incidence of 57.2% for the VOND in 1997 (P <.0001). Antenatal corticosteroids were given to 62.6% of infants in our center compared with 48.6% in the VOND (P <.005). In addition, 48.5% of our infants weighing less than 1500 g received oxygen at 36 weeks' postconceptional age versus 29.5% of the VOND infants (P <.001). Upon discharge to home, 37.5% of our infants were on oxygen compared with 15.6% of infants from all VOND centers, excluding the University of Kentucky (P <.001). CONCLUSION: The incidence of ROP in our center from 1995 to 1997 and in the VOND in 1997 show a significant decrease from the 65.8% incidence from 1986 to 1987 reported by the Multicenter Trial of Cryotherapy for ROP.

Ocean circulation off east Antarctica affects ecosystem structure and sea-ice extent.

Sea ice and oceanic boundaries have a dominant effect in structuring Antarctic marine ecosystems. Satellite imagery and historical data have identified the southern boundary of the Antarctic Circumpolar Current as a site of enhanced biological productivity. Meso-scale surveys off the Antarctic peninsula have related the abundances of Antarctic krill (Euphausia superba) and salps (Salpa thompsoni) to inter-annual variations in sea-ice extent. Here we have examined the ecosystem structure and oceanography spanning 3,500 km of the east Antarctic coastline, linking the scales of local surveys and global observations. Between 80 degrees and 150 degrees E there is a threefold variation in the extent of annual sea-ice cover, enabling us to examine the regional effects of sea ice and ocean circulation on biological productivity. Phytoplankton, primary productivity, Antarctic krill, whales and seabirds were concentrated where winter sea-ice extent is maximal, whereas salps were located where the sea-ice extent is minimal. We found enhanced biological activity south of the southern boundary of the Antarctic Circumpolar Current rather than in association with it. We propose that along this coastline ocean circulation determines both the sea-ice conditions and the level of biological productivity at all trophic levels.

A crystallizable form of RIIbeta regulatory domain obtained by limited proteolysis.

The type RIIbeta regulatory subunit of protein kinase A is primarily expressed in adipose tissue and brain. Knockout mice suggest a role for RIIbeta in regulating energy balance and adipose-tissue content, thus making it a potential target for therapeutic intervention in obesity. A truncated version of the RIalpha subunit has been used in a crystallographic study and was used here to design an analogous RIIbeta construct. Despite substantial screening, conditions were not found for the crystallization of the truncated RIIbeta subunit. However, limited proteolysis of the full-length RIIbeta subunit identified boundaries of the 'hinge' region and a fragment containing the two cAMP-binding domains which did crystallize. A recombinant version of the fragment was expressed and crystallized for X-ray diffraction studies. The crystals belong to the orthorhombic space group C222, with unit-cell parameters a = 91.6, b = 105.9, c = 85.8 A, and diffracted to at least 2.3 A.

Crystal structure of human squalene synthase. A key enzyme in cholesterol biosynthesis.

Squalene synthase catalyzes the biosynthesis of squalene, a key cholesterol precursor, through a reductive dimerization of two farnesyl diphosphate (FPP) molecules. The reaction is unique when compared with those of other FPP-utilizing enzymes and proceeds in two distinct steps, both of which involve the formation of carbocationic reaction intermediates. Because FPP is located at the final branch point in the isoprenoid biosynthesis pathway, its conversion to squalene through the action of squalene synthase represents the first committed step in the formation of cholesterol, making it an attractive target for therapeutic intervention. We have determined, for the first time, the crystal structures of recombinant human squalene synthase complexed with several different inhibitors. The structure shows that SQS is folded as a single domain, with a large channel in the middle of one face. The active sites of the two half-reactions catalyzed by the enzyme are located in the central channel, which is lined on both sides by conserved aspartate and arginine residues, which are known from mutagenesis experiments to be involved in FPP binding. One end of this channel is exposed to solvent, whereas the other end leads to a completely enclosed pocket surrounded by conserved hydrophobic residues. These observations, along with mutagenesis data identifying residues that affect substrate binding and activity, suggest that two molecules of FPP bind at one end of the channel, where the active center of the first half-reaction is located, and then the stable reaction intermediate moves into the deep pocket, where it is sequestered from solvent and the second half-reaction occurs. Five alpha helices surrounding the active center are structurally homologous to the active core in the three other isoprenoid biosynthetic enzymes whose crystal structures are known, even though there is no detectable sequence homology.

Adenylyl cyclase activity is increased in younger, but decreased in older, alcoholic patients after detoxification.

Acute and chronic administration of ethanol leads to alterations of the adenylyl cyclase (AC) signal transduction pathway. This study examined whether the formation of cAMP by AC in lymphocytes correlates with age in alcoholic patients and in healthy controls. Blood was drawn for preparation of lymphocyte membranes and for determination of basal, GTPgammaS-stimulated, and forskolin-stimulated AC activity from 68 actively drinking alcoholic patients (age, mean +/- SD: 45 +/- 10; range: 26-69 years) after ethanol detoxification. The patients' AC activity correlated negatively with age. In contrast, no effect of age was observed in the healthy controls (age, mean +/- SD: 42 +/- 11; range: 24-65 years). The age-related decrease in AC activity of alcoholic patients could not be attributed to the duration of regular alcohol intake. It was partly due to the large variance of AC activity in younger and middle-aged alcoholics.

Characterization of porcine T lymphocytes and their immune response against viral antigens.

T lymphocytes play a central role in the antigen-specific immune response against various pathogens. To detect and to characterize porcine T lymphocytes, monoclonal antibodies (mAb) against leukocyte differentiation antigens had been raised and classified for their specificity. Analyses of porcine T lymphocytes with specific mAb against CD4 and CD8 differentiation antigens revealed differences in the composition of the porcine T-lymphocyte population compared to other species. In addition to the known subpopulations, CD4+CD8- T helper cells and CD4-CD8+ cytolytic T lymphocytes, extra-thymic CD4+CD8+ T lymphocytes and a substantial proportion of CD2-CD4-CD8- T cell receptor (TcR)-gamma delta+ T cells could be detected in swine. Functional analyses of porcine T-lymphocyte subpopulations revealed the existence of two T-helper cell fractions with the phenotype CD4+CD8- and CD4+CD8+. Both were reactive in primary immune responses in vitro, whereas only cells derived from the CD4+CD8+ T-helper-cell subpopulation were able to respond to recall antigen in a secondary immune response. With regard to T lymphocytes with cytolytic activities, two subsets within the CD4-CD8+ T-cell subpopulation could be defined by the expression of CD6 differentiation antigens: CD6- cells which showed spontaneous cytolytic activity and CD6+ MHC I-restricted cytolytic T lymphocytes including virus-specific cytolytic T lymphocytes. These results enable now a detailed view into the porcine T-cell population and the reactivity of specific T cells involved in the porcine immune response against pathogens. Furthermore this knowledge offers the possibility to investigate specific interactions of porcine T lymphocytes with virus-specific epitopes during vaccination and viral infections.

A three-day course of dexamethasone therapy to prevent chronic lung disease in ventilated neonates: a randomized trial.

BACKGROUND: Although several trials of early dexamethasone therapy have been completed to determine if such therapy would reduce mortality and chronic lung disease (CLD) in infants with respiratory distress, optimal duration and side effects of such therapy remain unknown. PURPOSE: The purpose of this study was: 1) to determine if a 3-day course of early dexamethasone therapy would reduce CLD and increase survival without CLD in neonates who received surfactant therapy for respiratory distress syndrome and 2) to determine adverse effects associated with such therapy. DESIGN: This was a prospective multicenter randomized trial comparing a 3-day course of dexamethasone therapy beginning at 24 to 48 hours of life to placebo therapy. Two hundred forty-one neonates (dexamethasone n = 118, placebo n = 123), who weighed between 500 g and 1500 g, received surfactant therapy, and were at significant risk for CLD or death using a model to predict CLD or death at 24 hours of life, were enrolled in the trial. Infants randomized to receive early dexamethasone were given 6 doses of dexamethasone at 12-hour intervals beginning at 24 to 48 hours of life. The primary outcomes compared were survival without CLD and CLD. CLD was defined by the need for supplemental oxygen at the gestational age of 36 weeks. Complication rates and adverse effects of study drug therapy were also compared. RESULTS: Neonates randomized to early dexamethasone treatment were more likely to survive without CLD (RR: 1.3; 95% CI: 1.03, 1.7) and were less likely to develop CLD (RR: 0.6; CI: 0.3, 0. 98). Mortality rates were not significantly different. Subsequent dexamethasone therapy use was less in early dexamethasone-treated neonates (RR: 0.8; CI: 0.7, 0.96). Very early (

Blood ethanol levels and adenylyl cyclase activity in lymphocytes of alcoholic patients.

BACKGROUND: The adenylyl cyclase (AC) signal transduction pathway is a target of acute and chronic ethanol actions. This study examined whether AC activity in lymphocyte membranes of male alcoholic patients correlated with blood concentrations of ethanol. METHODS: Patients (n = 13; mean age: 40 +/- 8 years) were studied on the day of admission (day 0) and 2 days later under detoxification. Moreover, 13 age-matched male healthy controls (mean age 40 +/- 9 years) were included. Lymphocyte membranes were prepared by differential centrifugation whereby blood ethanol was washed out. As a measure of AC activity the formation of cyclic adenosine monophosphate (cAMP) from adenosine triphosphate was determined without (basal activity) and with stimulation of the second messenger system by the guanosine triphosphate (GTP) analogue GTP gamma S (20 mumol/L) via the G-protein or by forskolin (100 mumol/L) acting directly on the AC enzyme. RESULTS: On day 0, when ethanol blood concentrations were 38-100 mmol/L, we found a significant negative correlation between ethanol blood levels and stimulated AC activities. On day 2, the negative correlation with blood ethanol levels of day 0 had disappeared. CONCLUSIONS: The consumption of ethanol affects the AC system in lymphocytes of alcohol-dependent patients by a persistent effect on the cAMP forming enzyme.

Inhibition of MMP-1 and MMP-13 with phosphinic acids that exploit binding in the S2 pocket.

Through the use of empirical and computational methods, phosphinate-based inhibitors of MMP-1 and MMP-13 that bind into the S2 pocket of these enzymes were designed. The synthesis and testing of 2 suggested that binding was occurring as hypothesized. Structure determination of a co-crystal of 2 bound to the catalytic domain of MMP-1 confirmed the binding mode. Substituents binding into S2, S1', S2' and S3', were optimized yielding compounds with low double-digit nM IC50's against these enzymes.

[Phenotypic and functional characterization of porcine T-lymphocytes]. / Phänotypische und funktionelle Charakterisierung von T-Lymphozyten des Schweines.

Monoclonal antibodies (mAb) against leucocyte differentiation antigens have altered the way in which immunologists examine the immune system. These mAb allow to identify distinct surface molecules on leukocyte populations, by which these cells can be classified, isolated and studied for their functional properties. This review summarises the knowledge about differentiation antigens useful in the characterisation of porcine T-lymphocytes. Furthermore it focuses on several properties of porcine T-lymphocytes and T-lymphocyte subpopulations.

House staff supervision, workload, and experience in the neonatal intensive care unit: results of a national survey.

One hundred fifty-nine pediatric chief residents were surveyed regarding characteristics of the neonatal intensive care unit rotation for house staff at their institution. We documented substantial interinstitution variability in house staff NICU rotations in terms of number of rotations, and the workload and supervision of house staff.

Overview of the Second International Workshop to define swine cluster of differentiation (CD) antigens.

The aim of the Second International Swine Cluster of Differentiation (CD) Workshop, supported by the Veterinary Immunology Committee (VIC) of the International Union of Immunological Societies (IUIS), was to standardize the assignment of monoclonal antibodies (mAb) reactive with porcine leukocyte differentiation antigens and to define new antibody clusters. At the summary meeting of the workshop in July, 1995, revisions in the existing nomenclature for Swine CD were approved, so that the rules are now in accord with those for human and ruminant CD. Swine CD numbers will now be given to clusters of mAb to swine orthologues of human CD molecules when homology is proven by (1) suitable tissue distribution and lymphoid cell subset expression, (2) appropriate molecular mass of the antigen recognized by the mAbs, and (3) reactivity of mAbs with the cloned swine gene products, or cross-reactivity of the mAb on the human gene products. In some cases, this reactivity would not be fully proven, mainly due to the lack of cloned gene products; for these CD antigens, the respective clusters will be assigned by the prefix 'w' which will lead to 'wCD' antigens. As a result of the Second International Swine CD Workshop the assignment of 16 mAb to existing CD groups (CD2a, CD4a, CD5a, wCD6, wCD8, CD14, CD18a, wCD21, wCD25) was confirmed, and 2 mAb to existing swine workshop clusters (SWC). More importantly, for the work on the porcine immune system, was the definition of 5 new swine CD antigens, namely CD3 (recognized by 6 new mAb and 3 epitopes), CD16 (1 new mAb), wCD29 (2 mAb), CD45RA (3 mAb) and CD45RC (1 new mAb). Finally, the demarcation of two new SWC molecules in swine, SWC8 (2 mAb) and SWC9 (2 mAb) was confirmed.

Summary of the first round analyses of the Second International Swine CD Workshop.

The reactivity of 176 monoclonal antibodies (mAb) submitted to the Second International Swine CD Workshop, together with 19 internal standards, was analyzed by flow cytometry on 16 different cell types as a means of establishing the proper cell subset for later detailed clustering analyses. The exact CD subset reactivity of the 19 internal standard mAb had been characterized in the First International Swine CD Workshop. The flow cytometric analyses resulted in 40 data sets which were then subjected to statistical clustering using the Leukocyte Typing Database IV (LTDB4) software. As result of this work, 22 clusters were defined. After review of these results, panels of mAb from the defined first round clusters were assigned to cell subsets. The respective mAb in those first round clusters were then distributed to subset group researchers for further examination during the second round of the workshop.

Infection with classical swine fever virus: effects on phenotype and immune responsiveness of porcine T lymphocytes.

T lymphocytes obtained from pigs infected with a lethal dose of classical swine fever virus were analysed for phenotypic changes in the composition of T-cell subpopulations and for alterations in their immune responsiveness in vitro during the course of disease. Viral antigen detected in all subpopulations and the selective depletion of CD4- CD8- gamma/delta T cells showed that peripheral blood T lymphocytes were affected in the terminal stage (14-19 days post-infection) of classical swine fever whereas no implications for T lymphocytes were obvious during the first 10 days after infection. Furthermore, a depletion of CD1+ CD4+ CD8+ 'common thymocytes' was characteristic for the infected animals. Studies on immune functions of peripheral T lymphocytes revealed an abrogation of cellular immune responses as early as 3-5 days after infection and thus before detection of viral antigens in these cells. The data suggest that early immunosuppression represents a crucial event for the manifestation of classical swine fever.

Elevated circulating calcitonin gene-related peptide in umbilical cord and infant blood associated with maternal and neonatal sepsis and shock.

The role of the sensory neuropeptide calcitonin gene-related peptide (CGRP) was studied in preterm and term neonates with sepsis and shock. CGRP levels in blood were measured by RIA. The identity of immunoreactive CGRP (irCGRP) in adult and infant human blood was confirmed by reverse phase-HPLC. CGRP levels were analyzed in a total of 189 samples (95 from cord blood and 94 from neonates). The gestational ages ranged from 24 to 43 wk, and the birth weights ranged from 520 to 4445 g. Cord samples were collected immediately after delivery and infant blood samples were collected within 12 h of birth. Samples were coded, and the data were assigned to groups after determination of CGRP levels. There was a weight- and gestation-dependent increase in irCGRP in the newborn population. The direct correlation of circulating CGRP with ascending birth weight and gestation may have significance in the development of the fetus. Infants with and without certain complications were grouped in 500-g intervals. CGRP levels in cord blood were significantly elevated when certain stressful situations existed in the mother. These included culture-positive chorioamnionitis, placental abruption, and severe preeclampsia. There was a similar elevation in CGRP in patient blood in infants with culture-positive sepsis and/or shock with blood pressure <2 SD from the mean for corresponding gestation. CGRP levels did not differ between male and female infants and did not appear to be influenced by type of delivery (vaginal versus cesarean section). There was no significant difference in CGRP level between cord and patient blood in preterm neonates, but at term gestation cord blood levels were slightly higher than those in the patient blood. These results suggest that inflammation and hemodynamic imbalance (e.g. shock) are associated with increased in CGRP levels in the circulation in neonates. Future studies will focus on the biologic effects of elevated CGRP during neonatal complications and will examine the utility of CGRP measurement for diagnosis and treatment of disease in preterm infants.