Citations

The planarity of peptide bonds is an assumption that underlies decades of theoretical modeling of proteins. Peptide bonds strongly deviating from planarity are considered very rare features of protein structure that occur for functional reasons. Here, empirical analyses of atomic-resolution protein structures reveal that trans peptide groups can vary by more than 25° from planarity and that the true extent of nonplanarity is underestimated even in 1.2 Å resolution structures. Analyses as a function of the φ , ψ -backbone dihedral angles show that the expected value deviates by ± 8° from planar as a systematic function of conformation, but that the large majority of variation in planarity depends on tertiary effects. Furthermore, we show that those peptide bonds in proteins that are most nonplanar, deviating by over 20° from planarity, are not strongly associated with active sites. Instead, highly nonplanar peptides are simply integral components of protein structure related to local and tertiary structural features that tend to be conserved among homologs. To account for the systematic φ , ψ -dependent component of nonplanarity, we present a conformation-dependent library that can be used in crystallographic refinement and predictive protein modeling.

Abstract BACKGROUND Aberrantly methylated genes represent important markers for cancer diagnosis. We describe a multiplex detection approach to efficiently quantify these markers for clinical applications such as colorectal cancer screening. METHODS Quantitative allele-specific real-time target and signal amplification (QuARTS) combines a polymerase-based target amplification with an invasive cleavage-based signal amplification. The fluorescence signal is detected in a fashion similar to real-time PCR. We measured the dynamic range and analytical sensitivity of multiplex QuARTS reactions with titrated plasmid DNA. We used the QuARTS technology to quantify methylated BMP3, NDRG4, VIM, and TFPI2 genes on 91 DNA samples extracted from colorectal tissues, including 37 cancers, 25 adenomas, and 29 healthy epithelia. The assays were designed in triplex format that incorporated ACTB as a reference gene. Percent methylation was calculated by dividing methylated strands over ACTB strands and multiplying by 100. RESULTS The QuARTS method linearly detected methylated or unmethylated VIM gene down to 10 copies. No cross-reactivity was observed when methylated assays were used to amplify 105 copies of unmethylated gene and vice versa. The multiplex assay detected methylated genes spiked in unmethylated genes at a 0.01% ratio and vice versa. At a diagnostic specificity cutoff of 95%, methylated BMP3, NDRG4, VIM, and TFPI2 detected 84%, 92%, 86%, and 92% of colorectal cancers and 68%, 76%, 76%, and 88% of adenomas, respectively. CONCLUSIONS The QuARTS technology provides a promising approach for quantifying methylated markers. The markers assayed highly discriminated colorectal neoplasia from healthy epithelia.

Dietary sodium and blood pressure regulation differs between normotensive men and women, an effect which may involve endothelial production of nitric oxide (NO). Therefore, we tested the hypothesis that differences in the NO component of endothelium-dependent vasodilation between low and high dietary sodium intake depend on sex. For 5 days prior to study, healthy adults consumed a controlled low-sodium diet (10 mmol/day, n = 30, mean age ± SE: 30 ± 1 yr, 16 men) or high-sodium diet (400 mmol/day, n = 36, age 23 ± 1 yr, 13 men). Forearm blood flow (FBF, plethysmography) responses to brachial artery administration of acetylcholine (ACh, 4 μg·100 ml tissue−1·min−1) were measured before and after endothelial NO synthase inhibition with NG-monomethyl-l-arginine (l-NMMA, 50 mg bolus + 1 mg/min infusion). The NO component of endothelium-dependent dilation was calculated as the response to ACh before and after l-NMMA accounting for changes in baseline FBF: [(FBF ACh − FBF baseline) − (FBF AChL-NMMA − FBF baselineL-NMMA)]. This value was 5.7 ± 1.3 and 2.5 ± 0.8 ml·100 ml forearm tissue−1·min−1 for the low- and high-sodium diets, respectively (main effect of sodium, P = 0.019). The sodium effect was larger for the men, with values of 7.9 ± 2.0 and 2.2 ± 1.4 for men vs. 3.1 ± 1.3 and 2.7 ± 1.0 ml·100 ml forearm tissue−1·min−1 for the women ( P = 0.034, sex-by-sodium interaction). We conclude that the NO component of endothelium-dependent vasodilation is altered by dietary sodium intake based on sex, suggesting that endothelial NO production is sensitive to dietary sodium in healthy young men but not women.

Cilia are essential for normal development. The composition and assembly of cilia has been well characterized, but the signaling and transcriptional pathways that govern ciliogenesis remain poorly studied. Here, we report that Wnt/β-catenin signaling directly regulates ciliogenic transcription factor foxj1a expression and ciliogenesis in zebrafish Kupffer’s vesicle (KV). We show that Wnt signaling acts temporally and KV cell-autonomously to control left-right (LR) axis determination and ciliogenesis. Specifically, reduction of Wnt signaling leads to a disruption of LR patterning, shorter and fewer cilia, a loss of cilia motility and a downregulation of foxj1a expression. However, these phenotypes can be rescued by KV-targeted overexpression of foxj1a. In comparison to the FGF pathway that has been previously implicated in the control of ciliogenesis, our epistatic studies suggest a more downstream function of Wnt signaling in the regulation of foxj1a expression and ciliogenesis in KV. Importantly, enhancer analysis reveals that KV-specific expression of foxj1a requires the presence of putative Lef1/Tcf binding sites, indicating that Wnt signaling activates foxj1a transcription directly. We also find that impaired Wnt signaling leads to kidney cysts and otolith disorganization, which can be attributed to a loss of foxj1 expression and disrupted ciliogenesis in the developing pronephric ducts and otic vesicles. Together, our data reveal a novel role of Wnt/β-catenin signaling upstream of ciliogenesis, which might be a general developmental mechanism beyond KV. Moreover, our results also prompt a hypothesis that certain developmental effects of the Wnt/β-catenin pathway are due to the activation of Foxj1 and cilia formation.

The severity of preoperative cerebral palsy appears to correlate directly with postoperative complications. The primary aim of this study was to characterize the frequency of perioperative morbidity and mortality in cerebral palsy patients undergoing anesthesia. This was accomplished by undertaking a systematic review of the Mayo Database. The risk for perioperative adverse events was 63.1% (95% confidence interval 59.8%-66.5%). However, it deserves clarification that hypothermia and clinically significant yet non–life-threatening hypotension represented the majority (80%) of these complications. When these 2 events are excluded, the rate of adverse perioperative events was 13.1% (95% confidence interval 10.8%-15.5%). Risk factors associated with increased risk included American Society of Anesthesiologists physical status score exceeding 2, history of seizures, upper airway hypotonia, general surgery procedures, and adults. Our findings are useful to counsel patients with cerebral palsy, their caregivers, and their guardians regarding the risk of general anesthesia.