Experimental Measurement of Surface Charge Effects on the Stability of a Surface-Bound Biopolymer.

Quantitative experimental studies of the thermodynamics with which biopolymers interact with specific surfaces remain quite limited. In response, here we describe experimental and theoretical studies of the change in folding free energy that occurs when a simple biopolymer, a DNA stem-loop, is site-specifically attached to a range of chemically distinct surfaces generated via self-assembled monolayer formation on a gold electrode. Not surprisingly, the extent to which surface attachment alters the biopolymer's folding free energy depends strongly on the charge of the surface, with increasingly negatively charged surfaces leading to increased destabilization. A simple model that considers only the excluded volume and electrostatic repulsion generated by the surface and models the ionic environment above the surface as a continuum quantitatively recovers the observed free energy change associated with attachment to weakly charged negative surfaces. For more strongly charged negative surfaces a model taking into account the discrete size of the involved ions is required. Our studies thus highlight the important role that electrostatics can play in the physics of surface-biomolecule interactions.

Random coil negative control reproduces the discrepancy between scattering and FRET measurements of denatured protein dimensions.

Small-angle scattering studies generally indicate that the dimensions of unfolded single-domain proteins are independent (to within experimental uncertainty of a few percent) of denaturant concentration. In contrast, single-molecule FRET (smFRET) studies invariably suggest that protein unfolded states contract significantly as the denaturant concentration falls from high (∼6 M) to low (∼1 M). Here, we explore this discrepancy by using PEG to perform a hitherto absent negative control. This uncharged, highly hydrophilic polymer has been shown by multiple independent techniques to behave as a random coil in water, suggesting that it is unlikely to expand further on the addition of denaturant. Consistent with this observation, small-angle neutron scattering indicates that the dimensions of PEG are not significantly altered by the presence of either guanidine hydrochloride or urea. smFRET measurements on a PEG construct modified with the most commonly used FRET dye pair, however, produce denaturant-dependent changes in transfer efficiency similar to those seen for a number of unfolded proteins. Given the vastly different chemistries of PEG and unfolded proteins and the significant evidence that dye-free PEG is well-described as a denaturant-independent random coil, this similarity raises questions regarding the interpretation of smFRET data in terms of the hydrogen bond- or hydrophobically driven contraction of the unfolded state at low denaturant.

Using the population-shift mechanism to rationally introduce "Hill-type" cooperativity into a normally non-cooperative receptor.

Allosteric cooperativity, which nature uses to improve the sensitivity with which biomolecular receptors respond to small changes in ligand concentration, could likewise be of use in improving the responsiveness of artificial biosystems. Thus motivated, we demonstrate here the rational design of cooperative molecular beacons, a widely employed DNA sensor, using a generalizable population-shift approach in which we engineer receptors that equilibrate between a low-affinity state and a high-affinity state exposing two binding sites. Doing so we achieve cooperativity within error of ideal behavior, greatly steepening the beacon's binding curve relative to that of the parent receptor. The ability to rationally engineer cooperativity should prove useful in applications such as biosensors, synthetic biology and "smart" biomaterials, in which improved responsiveness is of value.

Effects of crowding on the stability of a surface-tethered biopolymer: an experimental study of folding in a highly crowded regime.

The high packing densities and fixed geometries with which biomolecules can be attached to macroscopic surfaces suggest that crowding effects may be particularly significant under these often densely packed conditions. Exploring this question experimentally, we report here the effects of crowding on the stability of a simple, surface-attached DNA stem-loop. We find that crowding by densely packed, folded biomolecules destabilizes our test-bed biomolecule by ~2 kJ/mol relative to the dilute (noninteracting) regime, an effect that presumably occurs due to steric and electrostatic repulsion arising from compact neighbors. Crowding by a dense brush of unfolded biomolecules, in contrast, enhances its stability by ~6 kJ/mol, presumably due to excluded volume and electrostatic effects that reduce the entropy of the unfolded state. Finally, crowding by like copies of the same biomolecule produces a significantly broader unfolding transition, likely because, under these circumstances, the stabilizing effects of crowding by unfolded molecules increase (and the destabilizing effects of neighboring folded molecules decrease) as more and more neighbors unfold. The crowding of surface-attached biomolecules may thus be a richer, more complex phenomenon than that seen in homogeneous solution.

Determinants of the detection limit and specificity of surface-based biosensors.

Here, we employ a model electrochemical DNA sensor to demonstrate that the detection limit and specificity of surface-based sensors often are not dependent on the true affinity of the probe for its target but are simply dependent on the effective probe concentration. Under these circumstances, the observed affinity (and thus the sensor's detection limit and specificity) will depend on the density with which the probes are packed on the surface of the sensor, the surface area, and even the volume of sample employed.

Entropic and electrostatic effects on the folding free energy of a surface-attached biomolecule: an experimental and theoretical study.

Surface-tethered biomolecules play key roles in many biological processes and biotechnologies. However, while the physical consequences of such surface attachment have seen significant theoretical study, to date this issue has seen relatively little experimental investigation. In response we present here a quantitative experimental and theoretical study of the extent to which attachment to a charged-but otherwise apparently inert-surface alters the folding free energy of a simple biomolecule. Specifically, we have measured the folding free energy of a DNA stem loop both in solution and when site-specifically attached to a negatively charged, hydroxylalkane-coated gold surface. We find that whereas surface attachment is destabilizing at low ionic strength, it becomes stabilizing at ionic strengths above ~130 mM. This behavior presumably reflects two competing mechanisms: excluded volume effects, which stabilize the folded conformation by reducing the entropy of the unfolded state, and electrostatics, which, at lower ionic strengths, destabilizes the more compact folded state via repulsion from the negatively charged surface. To test this hypothesis, we have employed existing theories of the electrostatics of surface-bound polyelectrolytes and the entropy of surface-bound polymers to model both effects. Despite lacking any fitted parameters, these theoretical models quantitatively fit our experimental results, suggesting that, for this system, current knowledge of both surface electrostatics and excluded volume effects is reasonably complete and accurate.

[Bupropion SR for weaning from smoking in relapsed smokers: results of an open multicentre trial in Germany]. / Bupropion SR als Unterstützung in der Raucherentwöhnung rückfälliger Raucher: Ergebnisse einer offenen multizentrischen Studie in Deutschland.

BACKGROUND: After initially successful smoking cessation the majority of patients relapse which stresses the addiction character of nicotine dependence. The patient and the physician, in therapeutic nihilism will not make another attempt for smoking cessation. In two randomised, double-blind, placebo-controlled trials in the US and Canada relapsed smokers, who had initially successfully quit smoking under Bupropion SR (Zyban), were again treated with Bupropion SR for smoking cessation. The encouraging results prompted us to perform a study in Germany under the conditions and temporal restraints of a routine medical practice. METHODS/PATIENTS: In this open multicentre study 321 patients of a total of 365 patients at 62 study sites were included. The patients represented a healthy population (= free of severe chronic disease) of smokers, who had relapsed after a smoking cessation attempt with Bupropion SR or nicotine replacement therapy and were currently smoking at least 20 cig./d for > 3 months. The treatment with Bupropion SR (300 mg/d) lasted 7 weeks, with initial run-in of 150 mg/d for the first three days; motivational counselling was provided during 4 clinical visits within the study duration of 26 weeks. RESULTS: 55.4 % of the patients were heavily or very heavily nicotine dependent as detected by the Fagerström Tolerance Questionnaire (score 6 - 11). The continuous abstinence rates for week 4 - 7 were 29.6 % (ITT) the point prevalence rates for week 26 were 30.5 % (ITT). Patients who showed a relapse had less daily cigarette consumption even after 26 weeks (10.3 cig./d versus 26.9 cig./d pretreatment). Nicotine craving reached a low plateau not earlier than week 6. Bupropion SR was well tolerated, only 9.7 % of the patients (ITT) withdrew from the study due to an adverse event. The most frequent adverse events were pain of the musculoskeletal system and insomnia. CONCLUSIONS: Bupropion SR was effective and well tolerated when given as an aid in smoking cessation in relapsed smokers.

Tocolytic magnesium sulfate toxicity and unexpected neonatal death.

A 31-year-old primigravida with twins had spontaneous rupture of the membranes at 32 weeks' gestation. On admission, because of contractions, the mother was started on tocolytic magnesium sulfate (MgSO4) along with betamethasone and prophylactic antibiotics. About a day later, she was found to have magnesium toxicity. Her serum total magnesium level was 9.0 mg/dl. Tocolysis was immediately discontinued. At cesarean delivery the following day, twin A, a female, died at 30 minutes of age despite a vigorous resuscitation. Although the preceding fetal heart rate patterns had been reassuring and the umbilical blood gases were normal, quite unexpectedly, the Apgar scores were 1/1/0. An autopsy revealed no anatomic abnormalities. Twin B, a female who survived, was also intubated at delivery. During her stay in the Neonatal Intensive Care Unit, she was found to have modestly elevated levels of serum cardiotroponin T. In our opinion, it is probable that the death of twin A can be directly attributed to magnesium sulfate toxicity. Neonatologists who attend deliveries should be aware that unexpected death may occur in babies who were exposed to high doses of tocolytic MgSO4.

Jaundice and breastfeeding.

Optimal management of breastfeeding does not eliminate neonatal jaundice and elevated serum bilirubin concentrations. Rather, it leads to a pattern of hyperbilirubinemia that is normal and, possibly, beneficial to infants. Excessive frequency of exaggerated jaundice in a hospital or community population of breastfed infants may be a warning that breastfeeding policies and support are not ideal for the establishment of good breastfeeding practices. The challenge to clinicians is to differentiate normal patterns of jaundice and hyperbilirubinemia from those that indicate an abnormality or place an infant at risk.

Low glucose-6-phosphate dehydrogenase enzyme activity level at the time of hemolysis in a male neonate with the African type of deficiency.

Glucose-6-phosphate dehydrogenase (G6PD) levels are not usually drawn in the evaluation of black neonates with hyperbilirubinemia because of the oft-stated opinion that the levels may be normal at the time of hemolysis and thus will be misleading. In fact, this opinion is not applicable to newborns as many studies have shown that deficiency in the conjugating ability of the liver, not hemolysis, is the main cause of neonatal jaundice associated with G6PD deficiency. We present a case report of a neonate with brisk hemolysis and hyperbilirubinemia in whom the G6PD level was abnormally low at the time of the hemolytic episode. DNA analysis showed him to have the A-(202A,376G) variant and, as well, the UGT1A1 promoter repeat polymorphism associated with Gilbert's disease. This case, as well as a review of the literature, indicates that enzyme levels are not normal in patients with G6PD A- who are undergoing hemolysis.

Role of naloxone in newborn resuscitation.

OBJECTIVE: Because of questions about the basis for the use of naloxone in resuscitation of the newborn, we wished to evaluate the use of naloxone at our institution and an affiliated hospital. METHODOLOGY: Evaluation of the actual use of naloxone at a university hospital and a community hospital: we document naloxone use by daily survey for a month in one; in the other, we perform a retrospective record review of 1 year's use. RESULTS: The university hospital had 240 births during February, 1998. Naloxone was given twice: once, 7 minutes before delivery to a woman at term who had received opiates about 2 hours previously; and once, intramuscularly, to a premature infant for apnea, before being intubated. The community hospital had 2044 births during fiscal 1998. Twenty-six neonates were identified as having received naloxone. Of the 26, 13 received naloxone without needing ventilatory support; all 13 with respiratory depression had a predisposing perinatal complication. CONCLUSION: The use of naloxone in practice may not conform to the American Academy of Pediatrics' guidelines for use in resuscitation of the newborn. The use of naloxone in resuscitation of the newborn should be reevaluated.

Significant ABO hemolytic disease of the newborn in a group B infant with a group A2 mother.

ABO hemolytic disease of the newborn (HDN) occurs almost exclusively in infants of blood group A or B who are born to group O mothers because IgG anti-A or -B occurs more commonly in group O than in group A or B individuals. We report a case in which clinically significant ABO-HDN occurred in a group B neonate from anti-B of a group A2 mother. The IgG anti-B titer was much higher (256) than that found in a group A1 mother/infant control group (

Length of pregnancy in African Americans: validation of a new predictive rule.

This study evaluated whether a new predictive rule is more accurate for estimating the length of pregnancy in African Americans than Nägele's rule, the accepted standard. After identifying women in early pregnancy, telephone interviews were conducted to obtain information about 16 previously established determinants of gestational length. Based on these data, a linear multivariate regression model was used to predict an estimated delivery date (EDD) for each mother. In addition, the EDD was determined using Nägele's rule. Later, the actual delivery date was compared with the EDD predicted by the new rule and with the EDD predicted by Nägele's rule. Each pregnancy was assigned to its better prediction group, either the new rule's group or the Nägele's rule group. Fifty-seven pregnancies were identified prospectively and monitored. The new rule predicted the actual delivery date more accurately in 66% (37/56) of pregnancies, Nägele's rule was a better predictor in 34% (19/56) of pregnancies, and both rules were equally accurate in predicting the delivery date for one pregnancy. The new rule was more precise than Nägele's rule (P = .022) when the binomial distribution was used. When using the linear regression model rule, a more accurate EDD can be determined for African-American women. Moreover, it is possible to predict the risk of preterm delivery (those occurring > 3 weeks earlier than the EDD).

Neonatal circumcision. Randomized trial of a sucrose pacifier for pain control.

OBJECTIVE: To assess the effectiveness of oral sucrose via a nipple compared with no treatment and dorsal penile nerve block (DPNB) for alleviating pain in neonatal circumcision. DESIGN: Randomized control trial. Data analysis performed by investigators blinded to the 3 treatment groups. SETTING: University teaching hospital, General Care Nursery. PATIENTS: One hundred nineteen full-term male, normal birth weight neonates, 12 hours old or older. INTERVENTIONS: No treatment (our standard care), DPNB, or oral sucrose prior to circumcision. MAIN OUTCOME MEASURES: Differences between groups in heart rate and oxygen saturation changes from baseline during specified intervals of the circumcision procedure. Differences between groups in loss of data due to episodes of excessive motion. RESULTS: Sucrose gave significant (P < .001) pain relief compared with the no treatment control throughout most of the circumcision and particularly in the early stages of the procedure. Overall, the average difference in the elevation of heart rates during the circumcision operative procedure among the 3 groups and the 95% confidence intervals (CIs) were as follows: control vs DPNB, 27.1 beats/min (17.6, 36.6) and control vs sucrose, 9.7 beats/min (0.1, 19.3). Furthermore, newborns who received either DPNB or sucrose had less loss of oxygen saturation data due to excessive motion during the procedure than the no-treatment controls. The total percentages of lost data due to excessive motion in the 3 groups were 31% for control, 10% for DPNB, and 8% for sucrose. Relative risk and 95% CIs were: DPNB vs control, 0.32 (0.23, 0.43); sucrose vs control, 0.26 (0.18, 0.36). Differences in oxygen saturation among the 3 groups during the circumcision operative procedure were statistically (P < .001), but perhaps not clinically, significant. However, the analysis did not include missing data due to excessive motion, which occurred predominantly in the no-treatment control group. CONCLUSION: Sucrose on a pacifier is an inexpensive and effective method for pain relief in neonatal circumcision when DPNB is not desirable.

Fetal death rate in the United States, 1979-1990: trend and racial disparity.

OBJECTIVE: To examine the impact of changes in birth weight distribution in individual groups and in birth weight-specific fetal death rates on the decline in the crude fetal death rate in the United States. METHODS: Data on live births and fetal deaths in the U.S. for the period 1979-1990 were examined by birth weight group and race using Kitagawa's method for analysis of the crude fetal death rate. RESULTS: In the period 1979-1990, all racial groups had a decrease in the crude fetal death rate, more so in whites and others (about 22%) than in blacks (10%). In the white population, 73.4% of the total reduction in the crude fetal death rate was attributable to the improvement in birth weight-specific fetal death rates, and the remaining portion of the reduction was due to a favorable change in birth weight distribution. In the black population, the reduction in the crude fetal death rate was entirely attributable to the improvement in the birth weight-specific fetal death rates. However, in other groups, a favorable change in the birth weight distribution was the major determinant. Although black births represented 16.5% of all births in the U.S., they accounted for 26-29% of the crude fetal death rate. Disparity in the crude fetal death rates for blacks and whites is explained almost entirely by differences in birth weight distribution. CONCLUSIONS: A further decrease in the crude fetal death rate in the U.S. requires a decrease in low birth weights, particularly in blacks.

Outcome of very low birth weight infants in industrialized countries: 1947-1987.

Neonatal intensive care has led to a progressive improvement in the survival of very low birth weight (VLBW, < 1,500 g) infants. However, it has not been established whether there has been a simultaneous increase or decrease in the prevalence of handicapping conditions in this group of children. To explore this question, a meta-analysis was performed using outcome data of 32 developmental studies of VLBW infants born in industrialized countries between 1947 and 1987. The authors' results show that the proportion of VLBW infants who survived and had an intact outcome progressively increased between 1947 and 1987--from 147 per 1,000 live births in the period 1947-1965 to 498 per 1,000 in the period 1980-1987 (p < 0.01). The prevalence of major handicapping conditions for the subset of VLBW infants who weighed < 1,000 g at birth increased, resulting from the increasing survival rates. However, the prevalence of major handicapping conditions among all children with VLBW decreased from 147 per 1,000 live births in 1947-1965 to 45 per 1,000 in 1980-1987 (p = 0.02). The authors' meta-analysis suggests that improved survival of VLBW infants has not been accompanied by an increase, but more likely a decrease, in the prevalence of handicapping conditions in this birth weight group.

Fetal death in a population of black women.

To describe the clinical causes of fetal death in black women, we performed a record review of the primary causes of fetal deaths (n = 315, > or = 500 g or > or = 24 weeks' gestation) occurring over an 11-year period in a population of 26,852 black women who delivered at the Chicago Lying-in Hospital, University of Chicago Hospitals, Chicago, IL. The over-all fetal death rate (FDR) per 1,000 total births was 11.7, consistent with U.S. vital statistics data for blacks. The FDR per 10,000 births attributed to hypertension was nine times greater in our population than in a historical comparison population of Canadian white women: 19.5 (95% CI = 13.7, 25.4) versus 2.2 (P < .0001), respectively, although the prevalence of hypertension was only 1.2 times greater in the population of black women. Furthermore, hypertension in pregnancy accounted for 15% of the excess fetal mortality in our population of urban black women as compared to the population of Canadian white women. Health care providers should be aware of the risk of fetal death in hypertensive, innercity, U.S. black women.