Vinod K. Misra

Single-molecule transition-state analysis of RNA folding

How RNA molecules fold into functional structures is a problem of great significance given the expanding list of essential cellular RNA enzymes and the increasing number of applications of RNA in biotechnology and medicine. A critical step toward solving the RNA folding problem is the characterization of the associated transition states. This is a challenging task in part because the rugged energy landscape of RNA often leads to the coexistence of multiple distinct structural transitions. Here, we exploit single-molecule fluorescence spectroscopy to follow in real time the equilibrium transitions between conformational states of a model RNA enzyme, the hairpin ribozyme. We clearly distinguish structural transitions between effectively noninterchanging sets of unfolded and folded states and characterize key factors defining the transition state of an elementary folding reaction where the hairpin ribozymes two helical domains dock to make several tertiary contacts. Our single-molecule experiments in conjunction with site-specific mutations and metal ion titrations show that the two RNA domains are in a contact or close-to-contact configuration in the transition state even though the native tertiary contacts are at most partially formed. Such a compact transition state without well formed tertiary contacts may be a general property of elementary RNA folding reactions.

A thermodynamic framework for Mg2+ binding to RNA

We present a model describing how Mg2+ binds and stabilizes specific RNA structures. In this model, RNA stabilization arises from two energetically distinct modes of Mg2+ binding: diffuse- and site-binding. Diffusely bound Mg2+ are electrostatically attracted to the strong anionic field around the RNA and are accurately described by the Poisson–Boltzmann equation as an ensemble distributed according to the electrostatic potentials around the nucleic acid. Site-bound Mg2+ are strongly attracted to specifically arranged electronegative ligands that desolvate the ion and the RNA binding site. Thus, site-binding is a competition between the strong coulombic attraction and the large cost of desolvating the ion and its binding pocket. By using this framework, we analyze three systems where a single site-bound Mg2+ may be important for stability: the P5 helix and the P5b stem loop from the P4-P6 domain of the Tetrahymena thermophila group I intron and a 58-nt fragment of the Escherichia coli 23S ribosomal RNA. Diffusely bound Mg2+ play a dominant role in stabilizing these RNA structures. These ions stabilize the folded structures, in part, by accumulating in regions of high negative electrostatic potential. These regions of Mg2+ localization correspond to ions that are observed in the x-ray crystallographic and NMR structures of the RNA. In contrast, the contribution of site-binding to RNA stability is often quite small because of the large desolvation penalty. However, in special cases, site-binding of partially dehydrated Mg2+ to locations with extraordinarily high electrostatic potential can also help stabilize folded RNA structures.

Maternal Serum Lipids During Pregnancy and Infant Birth Weight: The Influence of Prepregnancy BMI

Maternal obesity may be associated with metabolic factors that affect the intrauterine environment, fetal growth, and the offsprings long‐term risk for chronic disease. Among these factors, maternal serum lipids play a particularly important role. Our objective was to estimate the influence of variation in maternal serum lipid levels on variation in infant birth weight (BW) in overweight/obese and normal weight women. In a prospective cohort of 143 gravidas, we measured maternal serum levels of total cholesterol (TC), low‐density lipoprotein cholesterol (LDL‐C), high‐density lipoprotein cholesterol (HDL‐C), and triglycerides (TG) at 6–10, 10–14, 16–20, 22–26, and 32–36 weeks gestation. Effects of maternal serum lipid levels on infant BW adjusted for gestational age at delivery (aBW) were analyzed using linear regression models. In analyses stratified by maternal prepregnancy BMI categorized as normal (≤25.0 kg/m2) and overweight/obese (>25.0 kg/m2), we found a significant (P < 0.05) inverse association between aBW and HDL‐C at all time points starting at 10 weeks gestation in overweight/obese women. No significant effect was found in normal weight women. In contrast, increased maternal serum TG was significantly associated with increased aBW only for normal weight women at 10–14 and 22–26 weeks gestation. Variation in aBW is not associated with variation in maternal serum TC or LDL‐C for either stratum at any time point. We postulate that such differences may be involved in the “physiological programming” that influences later risk of chronic disease in the infants of overweight/obese mothers.

High Incidence of Noonan Syndrome Features Including Short Stature and Pulmonic Stenosis in Patients carrying NF1 Missense Mutations Affecting p.Arg1809: Genotype–Phenotype Correlation

Neurofibromatosis type 1 (NF1) is one of the most frequent genetic disorders, affecting 1:3,000 worldwide. Identification of genotype–phenotype correlations is challenging because of the wide range clinical variability, the progressive nature of the disorder, and extreme diversity of the mutational spectrum. We report 136 individuals with a distinct phenotype carrying one of five different NF1 missense mutations affecting p.Arg1809. Patients presented with multiple café‐au‐lait macules (CALM) with or without freckling and Lisch nodules, but no externally visible plexiform neurofibromas or clear cutaneous neurofibromas were found. About 25% of the individuals had Noonan‐like features. Pulmonic stenosis and short stature were significantly more prevalent compared with classic cohorts (P < 0.0001). Developmental delays and/or learning disabilities were reported in over 50% of patients. Melanocytes cultured from a CALM in a segmental NF1‐patient showed two different somatic NF1 mutations, p.Arg1809Cys and a multi‐exon deletion, providing genetic evidence that p.Arg1809Cys is a loss‐of‐function mutation in the melanocytes and causes a pigmentary phenotype. Constitutional missense mutations at p.Arg1809 affect 1.23% of unrelated NF1 probands in the UAB cohort, therefore this specific NF1 genotype–phenotype correlation will affect counseling and management of a significant number of patients.

Prepregnancy body mass index and gestational age-dependent changes in lipid levels during pregnancy.

OBJECTIVE: To examine the effect of maternal prepregnancy overweight and obesity on gestational age-dependent variation in lipid levels during pregnancy. METHODS: Women between 6 and 10 weeks of gestation who carry a single, live intrauterine pregnancy were eligible to participate in a prospective pregnancy study (N=142). The exposure, maternal prepregnancy body mass index (BMI), was classified as: normal weight (BMI 18.5–26.0 kg/m2) and overweight or obese (BMI over 26.0 kg/m2). Our outcomes of interest, total cholesterol, triglycerides, low-density lipoprotein (LDL) cholesterol, and high-density lipoprotein (HDL) cholesterol were measured at 6–10, 10–14, 16–20, 22–26, and 32–36 weeks of gestation. Mixed linear models were used to examine how the trajectory of lipid levels during pregnancy differs between overweight or obese and normal-weight women. RESULTS: Levels of total cholesterol, triglycerides, LDL cholesterol, and HDL choloesterol increased over the prenatal period. The rate of change in LDL cholesterol (P<.001) and total cholesterol (P=.01) levels was lower for overweight or obese women than for normal-weight women in late-second and early-third trimester. As a result, overweight or obese women had significantly lower total cholesterol and LDL cholesterol levels than their normal-weight counterparts between 32–36 weeks of gestation. CONCLUSION: Overweight and obese women have different lipid profiles during pregnancy than their normal-weight peers. This difference may be the result of metabolic dysregulation associated with maternal overweight and obesity that mediates the increased risk of adverse outcomes found in these women. LEVEL OF EVIDENCE: II

Contribution of the Closing Base Pair to Exceptional Stability in RNA Tetraloops: Roles for Molecular Mimicry and Electrostatic Factors

Hairpins are common RNA secondary structures that play multiple roles in nature. Tetraloops are the most frequent RNA hairpin loops and are often phylogenetically conserved. For both the UNCG and GNRA families, CG closing base pairs (cbps) confer exceptional thermodynamic stability but the molecular basis for this has remained unclear. We propose that, despite having very different overall folds, these two tetraloop families achieve stability by presenting the same functionalities to the major groove edge of the CG cbp. Thermodynamic contributions of this molecular mimicry were investigated using substitutions at the nucleobase and functional group levels. By either interrupting or deleting loop-cbp electrostatic interactions, which were identified by solving the nonlinear Poisson-Boltzmann (NLPB) equation, stability changed in a manner consistent with molecular mimicry. We also observed a linear relationship between DeltaG(o)(37) and log[Na(+)] for both families, and loops with a CG cbp had a decreased dependence of stability on salt. NLPB calculations revealed that, for both UUCG and GAAA tetraloops, the GC cbp form has a higher surface charge density, although it arises from changes in loop compaction for UUCG and changes in loop configuration for GAAA. Higher surface charge density leads to stronger interactions of GC cbp loops with solvent and salt, which explains the correlation between experimental and calculated trends of free energy with salt. Molecular mimicry as evidenced in these two stable but otherwise unrelated tetraloops may underlie common functional roles in other RNA and DNA motifs.

Placental Blood Flow and the Risk of Preterm Delivery

The goal of this analysis was to estimate the influence of variation in uterine artery and umbilical artery resistance indices (RIs) measured across gestation on variation in the risk of preterm delivery (PTD). Analyses were carried out on data collected in a longitudinal study of 523 gravidas. Uterine and umbilical artery RIs were measured on three occasions during pregnancy (16-20 weeks gestation; 21-29 weeks gestation; and 30-36 weeks gestation). Data were analyzed using the Cox proportional hazards regression model. The primary outcome variable was birth prior to 37 weeks gestation. We found that for mothers who delivered preterm the mean uterine artery RI was consistently larger across all gestational ages, while the mean umbilical artery RI decreased significantly more slowly across gestation than for their term counterparts. In analyses pooled by type of delivery, we found that the hazard ratio (HR) for PTD was statistically significant for either uterine artery RI (HR=2.26, 95% CI: 1.65, 3.11) or umbilical artery RI (HR=3.47, 95% CI: 2.43, 4.95) after adjusting for statistically significant covariates. In stratified analyses, the hazard ratio for PTD was also positively associated with an increased uterine or umbilical artery RI in both spontaneous and indicated deliveries. Our data suggest that pregnancies with either a higher uterine or umbilical artery RI across gestation are more likely to be affected by PTD suggesting that disordered placentation resulting in compromised placental blood flow may be an important pathway to PTD.

Maternal Serum Leptin During Pregnancy and Infant Birth Weight: the Influence of Maternal Overweight and Obesity

Few studies have examined whether the distinct metabolic patterns found in obese and nonobese pregnant women have different effects on the growing fetus. Our objective was to estimate the influence of longitudinal variation in maternal serum leptin levels on variation in infant birth weight in overweight/obese versus normal‐weight women.

The Influence of Overweight and Obesity on Maternal Soluble fms-Like Tyrosine Kinase 1 and Its Relationship With Leptin During Pregnancy

We studied obesity-related differences in the relation of maternal levels of leptin to levels of soluble fms-like tyrosine kinase 1 (sFlt1), an antiangiogenic protein that influences placentation and risk of adverse pregnancy outcomes. In a prospective cohort of 286 gravidas, we measured maternal serum levels of sFlt1 and leptin at 5 time points across pregnancy. Analyses stratified on prepregnancy body mass index (<25 vs ≥25) were done using mixed linear models. The mean leptin concentrations were significantly higher in overweight/obese compared to normal-weight women, while mean sFlt1 levels in second and third trimester were significantly higher in normal weight compared to overweight/obese women. The relationship between sFlt1 and leptin differed between the 2 strata. After controlling for maternal weight, a 1 ng/mL increase in leptin was associated with an 19.4 pg/mL increase in sFlt1 (P = .01) in normal-weight women, while leptin was not associated with sFlt1 (β = 1.1, P = .75) in overweight/obese women. Such differences suggest that metabolic differences in overweight/obese women compared to their normal weight peers may differentially impact the physiologic changes during pregnancy.

The effects of maternal weight gain patterns on term birth weight in African-American women

Objective. The goals of our study were (1) to estimate the trends in maternal weight gain patterns and (2) to estimate the influence of variation in maternal weight and rate of weight gain over different time periods in gestation on variation in birth weight in African-American and non-African-American gravidas. Study Design and Setting. Data from a prospective cohort study in which pregnant women were monitored at multiple time points during pregnancy were analysed. Maternal weight was measured at three times during pregnancy: preconception (W0); 16–20 weeks gestation (W1); 30–36 weeks gestation (W2), in a cohort of 435 women with full-term singleton pregnancies. The relationship between gestational age-adjusted birth weight (aBW) and measures of maternal weight and rate of weight gain across pregnancy was estimated using a multivariable longitudinal regression analysis stratified on African-American race. Results. The aBW was significantly associated with maternal weight measured at any visit in both strata. For African-American women, variation in aBW was significantly associated with variation in the rate of maternal weight gain in the first half of pregnancy (W01) but not the rate of maternal weight gain in the second half of pregnancy (W12); while for non-African-American women, variation in aBW was significantly associated with W12 but not W01. Conclusion. Factors influencing the relationship between aBW and maternal weight gain patterns depend on the context of the pregnancy defined by race. Clinical decisions and recommendations about maternal weight and weight gain during pregnancy may need to account for such heterogeneity.