Amnio acid substitution at position 298 of human glucose-6 phosphatase-α significantly impacts its stability in mammalian cells.

Glucose-6-phosphatase-α (G6Pase-α) catalyzes the hydrolysis of glucose-6-phosphate to glucose and functions as a key regulator in maintaining blood glucose homeostasis. Deficiency in G6Pase-α causes glycogen storage disease 1a (GSD1a), an inherited disorder characterized by life-threatening hypoglycemia and other long-term complications. We have developed a potential mRNA-based therapy for GSD1a and demonstrated that a human G6Pase-α (hG6Pase-α) variant harboring a single serine (S) to cysteine (C) substitution at the amino acid site 298 (S298C) had > twofold increase in protein expression, resulting in improved in vivo efficacy. Here, we sought to investigate the mechanisms contributing to the increased expression of the S298C variant. Mutagenesis of hG6Pase-α identified distinct protein variants at the 298 amino acid position with substantial reduction in protein expression in cultured cells. Kinetic analysis of expression and subcellular localization in mammalian cells, combined with cell-free in vitro translation assays, revealed that altered protein expression stemmed from differences in cellular protein stability rather than biosynthetic rates. Site-specific mutagenesis studies targeting other cysteines of the hG6Pase-α S298C variant suggest the observed improvements in stability are not due to additional disulfide bond formation. The glycosylation at Asparagine (N)-96 is critical in maintaining enzymatic activity and mutations at position 298 mainly affected glycosylated forms of hG6Pase-α. Finally, proteasome inhibition by lactacystin improved expression levels of unstable hG6Pase-α variants. Taken together, these data uncover a critical role for a single amino acid substitution impacting the stability of G6Pase-α and provide insights into the molecular genetics of GSD1a and protein engineering for therapeutic development.

Global patterns in price elasticities of sugar-sweetened beverage intake and potential effectiveness of tax policy: a cross-sectional study of 164 countries by sex, age and global-income decile.

OBJECTIVE: To quantify global relationships between sugar-sweetened beverage (SSB) intake and prices and examine the potential effectiveness of tax policy. DESIGN: SSB intake data by country, age and sex from the Global Dietary Database were combined with gross domestic product and price data from the World Bank. Intake responsiveness to income and prices was estimated accounting for national income, age and sex differences. SETTING: 164 countries. POPULATION: Full adult population in each country. MAIN OUTCOME MEASURES: A consumer demand modelling framework was used to estimate the relationship between SSB intake and prices and derive own-price elasticities (measures of percentage changes in intake from a 1% price change) globally by age and sex. We simulated how a 20% tax would impact SSB intake globally. Tax policy outcomes were examined across countries by global income decile for representative age and sex subgroups. RESULTS: Own-price responsiveness was highest in lowest income countries, ranging from -0.70 (p<0.100) for women, age 50, to -1.91 (p<0.001) for men, age 80. In the highest income countries, responsiveness was as high as -0.49 (p<0.001) (men, age 20), but was mostly insignificant for older adults. Overall, elasticities were strongest (more negative) at the youngest and oldest age groups, and mostly insignificant for middle-aged adults, particularly in middle-income and high-income countries. Sex differences were mostly negligible. Potential intake reductions from a 20% tax in lowest income countries ranged from 14.5% (95% CI: 29.5%, -0.4%) in women, 35 ≤ age < 60, to 24.9% (44.4%, 5.3%) in men, age ≥60. Intake reductions decreased with country income overall, and were mostly insignificant for middle-aged adults. CONCLUSIONS: These findings estimate the global price-responsiveness of SSB intake by age and sex, informing ongoing policy discussions on potential effects of taxes.

An empiric antibiotic protocol using risk stratification improves antibiotic selection and timing in critically ill children.

RATIONALE: Timely and appropriate empiric antibiotics can improve outcomes in critically ill patients with infection. Evidence and guidelines to guide empiric antibiotic decisions are lacking for critically ill children. OBJECTIVES: To evaluate the impact of an empiric antibiotic protocol on appropriateness of initial antibiotics and time to appropriate antibiotics in critically ill children with suspected infection. METHODS: A computer order entry-based, pediatric intensive care unit-specific, empiric antibiotic protocol including risk stratification for healthcare-associated infections was implemented in a tertiary pediatric intensive care unit. Antibiotic and culture data were evaluated for a total of 556 infectious episodes in 491 patients from 2004 (preprotocol, n = 252) and 2007 (protocol, n = 304) with suspected infection. Antibiotics appropriateness based on risk factors and culture results was assessed, as was time from initial culture to appropriate antibiotics. MEASUREMENTS AND MAIN RESULTS: Patients treated using the protocols were more likely to receive appropriate empiric antibiotics based on risk factors (76 vs. 15%; P < 0.0001) and culture results (89 vs. 64%; P < 0.0001). Patients treated after protocol implementation had a shorter time to appropriate antibiotics (median, 5.9 vs. 9.6 h; P < 0.0001), particularly in those who grew healthcare-associated pathogens (5.8 vs. 24 h; P = 0.0001). No significant baseline characteristic differences were seen. CONCLUSIONS: An empiric antibiotic protocol in the pediatric intensive care unit incorporating risk stratification for healthcare-associated infections resulted in increased appropriateness of empiric antibiotics and in decreased time to appropriate antibiotics in critically ill children with infection.

Monodisperse 130 kDa and 260 kDa Recombinant Human Hemoglobin Polymers as Scaffolds for Protein Engineering of Hemoglobin-Based Oxygen Carriers.

A recombinant 130 kDa dihemoglobin which is made up of a single-chain tetra-α globin and four ß globins has been expressed as a soluble protein in E. coli. The sequence of the single chain tetra-α is: αI-Gly-αII-(SerGlyGly)5Ser-αIII-Gly-αIV. This dihemoglobin has been purified and characterized in vitro by size exclusion chromatography, electrospray mass spectroscopy, equilibrium oxygen binding, and analytical ultracentrifugation. The observed values of P50 and nmax for the dihemoglobin are slightly lower than those observed for the recombinant hemoglobin rHb1.1 (a "monohemoglobin" comprised of two ß globins and an αI-Gly-αII diα-globin chain). Titration of the deoxy form of dihemoglobin with CO shows that all eight heme centers bind ligand. In vivo, dihemoglobin showed increased circulating halflife and a reduced pressor response in conscious rats when compared to rHb1.1. These observations suggest that dihemoglobin is an oxygen carrying molecule with desirable in vivo properties and provides a platform for an isooncotic hemoglobin solution derived solely from a recombinant source. A 260 kDa tetrahemoglobin has also been produced by chemical crosslinking of a dihemoglobin that contains a Lys16Cys mutation in the C-terminal α-globin subunit. Tetrahemoglobin also shows reduced vasoactivity in conscious rats that is comparable to that observed for dihemoglobin.

Induction of G-quadruplex DNA structure by Zn(II) 5,10,15,20-tetrakis(N-methyl-4-pyridyl)porphyrin.

G-quadruplexes (GQ) are formed by the association of guanine-rich stretches of DNA. Certain small molecules can influence kinetics and thermodynamics of this association. Understanding the mechanism of ligand-assisted GQ folding is necessary for the design of more efficient cancer therapeutics. The oligonucleotide d(TAGGG)(2) forms parallel bimolecular GQ in the presence of ≥66 mM K(+); GQs are not formed under Na(+), Li(+) or low K(+) conditions. The thermodynamic parameters for GQ folding at 60 µM oligonucleotide and 100 mM KCl are ΔH = -35 ± 2 kcal mol(-1) and ΔG(310) = -1.4 kcal mol(-1). Quadruplex [d(TAGGG)(2)](2) binds 2-3 K(+) ions with K(d) of 0.5 ± 0.2 mM. Our work addresses the question of whether metal free 5,10,15,20-tetrakis(N-methyl-4-pyridyl)porphyrin (TMPyP4) and its Zn(II), Cu(II), and Pt(II) derivatives are capable of facilitating GQ folding of d(TAGGG)(2) from single stranded, or binding to preformed GQ, using UV-vis and circular dichroism (CD) spectroscopies. ZnTMPyP4 is unique among other porphyrins in its ability to induce GQ structure of d(TAGGG)(2), which also requires at least a low amount of potassium. ZnTMPyP4 binds with 2:1 stoichiometry possibly in an end-stacking mode with a ~10(6) M(-1) binding constant, determined through UV-vis and ITC titrations. This process is entropically driven and has ΔG(298) of -8.0 kcal mol(-1). TMPyP4 binds with 3:1 stoichiometry and K(a) of ~10(6) M(-1). ZnTMPyP4 and TMPyP4 are efficient stabilizers of [d(TAGGG)(2)](2) displaying ΔT(1/2) of 13.5 and 13.8 °C, respectively, at 1:2 GQ to porphyrin ratio; CuTMPyP4 shows a much weaker effect (ΔT(1/2) = 4.7 °C) and PtTMPyP4 is weakly destabilizing (ΔT(1/2) = -2.9 °C). The selectivity of ZnTMPyP4 for GQ versus dsDNA is comparable to that of TMPyP4. The ability of ZnTMPyP4 to bind and stabilize GQ, to induce GQ formation, and speed up its folding may suggest an important biological activity for this molecule.

Timing of correct parenteral antibiotic initiation and outcomes from severe bacterial community-acquired pneumonia in children.

BACKGROUND: The impact of timing of appropriate antibiotic initiation for critically ill children with severe bacterial community-acquired pneumonia (CAP) is unknown. We hypothesized that longer time to initiation of correct parenteral antibiotic would be associated with longer durations of mechanical ventilation, intensive care unit length of stay, and hospital length of stay. METHODS: We retrospectively reviewed medical records of children admitted to Nationwide Children's Hospital between January 2004 and December 2006 with bacterial CAP treated with mechanical ventilation, excluding those with documented viral infection. Time to correct antibiotic was defined as time from presentation to any emergency department to the initiation of a parenteral antibiotic to which cultured pathogens were susceptible. RESULTS: In all, 45 patients, median age 17 months, were identified. Median time to correct antibiotic was 10.3 hours, with 71% of patients receiving correct empiric therapy. After adjusting for severity of illness, longer time to correct antibiotic was independently associated with longer hospital stay (P = 0.007). For the 23 patients in the cohort for whom pneumonia was the primary diagnosis, longer time to correct antibiotic was independently associated with longer durations of mechanical ventilation (P = 0.01), intensive care unit stay (P = 0.001), and hospital stay (P = 0.006). Delays in antibiotic administration as short as 2 to 4 hours were associated with adverse outcomes in this group. CONCLUSIONS: In our critically ill children with severe bacterial CAP, longer delays in receipt of appropriate empiric antibiotics were independently associated with adverse outcomes.

Failure to recover somatotropic axis function is associated with mortality from pediatric sepsis-induced multiple organ dysfunction syndrome.

OBJECTIVES: To describe the associations between mediators of the somatotropic axis and mortality from sepsis-induced multiple organ dysfunction syndrome in children; and to examine the relationship between immune function and the somatotropic axis in this setting. DESIGN: Retrospective study using banked plasma. SETTING: Single mixed surgical/medical intensive care unit at a quaternary level children's hospital. PATIENTS: A total of 24 children (n = 17 survivors, 7 nonsurvivors) with severe sepsis or septic shock and dysfunction of >or=2 organ systems. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Plasma samples were available from days 3, 7, and 14 of multiple organ dysfunction syndrome. Insulin-like growth factor 1 and insulin-like growth factor binding protein 3 levels were measured by chemiluminescence. Immune function was quantified using previously determined ex vivo lipopolysaccharide-induced tumor necrosis factor-alpha production levels and absolute lymphocyte counts. Insulin-like growth factor 1 levels were lower in nonsurvivors compared with survivors (p = .004) with the greatest difference seen on day 14 (25 [25-69] ng/mL vs. 314 [123-582] ng/mL; p = .038). insulin-like growth factor binding protein 3 levels were reduced similarly over time (p = .019). A drop in plasma insulin-like growth factor binding protein 3 level at any time after day 3 of illness resulted in a 35-fold increased odds of death (odds ratio, 35 [1.6-750]; p = .004). Both ex vivo tumor necrosis factor-alpha and absolute lymphocyte count were reduced in nonsurvivors compared with survivors, but these differences occurred earlier (days 3 and 7). CONCLUSIONS: These data suggest that prolonged reduction of somatotropic axis function is associated with mortality from pediatric sepsis-induced multiple organ dysfunction syndrome. Reductions in innate and adaptive immune function are common in this population and are associated with failure of recovery of the somatotropic axis, although the nature of these relationships remains incompletely understood.