Phagocytic function of polymorphonuclear leukocytes and the RES in endotoxemia

The reticuloendothelial system (RES) and the polymorphonuclear leukocytes (PMNs) are thought to play a major role in defense against sepsis. Disturbances in the function of these two phagocytic systems during a septic event is associated with the development of lung capillary injury. Endotoxemia is said to lead to similar changes. Our study examined the function of the RES and PMNs after bolus injection of endotoxin (2 micrograms/kg BW) in a standardized sheep model. For up to 24 hr after endotoxin, blood samples were drawn and PMN function was followed by chemiluminescence, chemotaxis, and adherence as well as the phagocytosis and killing of bacteria. RES function was determined by the blood clearance of a labeled Tc99 colloid. We found an increase of RES clearance directly after endotoxin. Chemotaxis, phagocytosis, and killing were reduced. Adherence was increased. Chemiluminescence peak maximum (CLPM), representing the metabolic activity of the PMNs, was initially increased but shortly thereafter showed a significant decline (at 1 hr: 0.52 +/- 0.13 X 10(6) cpm with P less than 0.05 compared to baseline). The chemiluminescence peak time (CLPT), a measure of membrane receptor function, was significantly reduced (10.0 +/- 2.2 min with P less than 0.001 compared to baseline). Endotoxin led to a reduction of intracellular PMN functions (phagocytosis, killing, CLPM) within 1 hr. Membrane localized functions (adherence, CLPT) were increased. The changes in PMN function might be the reason for the development of lung capillary injury, in spite of undisturbed RES clearance.     

A rapid method for estimating weight and resuscitation drug dosages from length in the pediatric age group

Drug dosages used during pediatric emergencies and resuscitation are often based on estimated body weight. The Broselow Tape, a tape measure that estimates weight and drug dosages for pediatric patients from their length, has been developed to facilitate proper dosing during emergencies. In our study, 937 children of known weight were measured with this tape. Weight estimates generated by the tape were found to be within 15% error for 79% of the children. The tape was found to be extremely accurate for children from 3.5 to 10 kg, and from 10 to 25 kg. Regression lines of estimated compared with actual weight for these children have slopes of 0.98 and 0.96, respectively, not significantly different from the ideal slope of 1.00 (P = 28 and .13). Accuracy was significantly decreased for measured children who weighed more than 25 kg. In a separate group of children (n = 53), the tape was shown to be more accurate than weight estimates made by residents and pediatric nurses (P less than .0001). Use of the Broselow Tape is a simple, accurate method of estimating pediatric weights and drug doses and eliminates the need for memorization and calculation.     

CADASIL mutations sensitize the brain to ischemia via spreading depolarizations and abnormal extracellular potassium homeostasis

Cerebral autosomal dominant arteriopathy, subcortical infarcts, and leukoencephalopathy (CADASIL) is the most common monogenic form of small vessel disease characterized by migraine with aura, leukoaraiosis, strokes, and dementia. CADASIL mutations cause cerebrovascular dysfunction in both animal models and humans. Here, we showed that 2 different human CADASIL mutations (Notch3 R90C or R169C) worsen ischemic stroke outcomes in transgenic mice; this was explained by the higher blood flow threshold to maintain tissue viability compared with that in wild type (WT) mice. Both mutants developed larger infarcts and worse neurological deficits compared with WT mice, regardless of age or sex after filament middle cerebral artery occlusion. However, full-field laser speckle flowmetry during distal middle cerebral artery occlusion showed comparable perfusion deficits in mutants and their respective WT controls. Circle of Willis anatomy and pial collateralization also did not differ among the genotypes. In contrast, mutants had a higher cerebral blood flow threshold, below which infarction ensued, suggesting increased sensitivity of brain tissue to ischemia. Electrophysiological recordings revealed a 1.5- to 2-fold higher frequency of peri-infarct spreading depolarizations in CADASIL mutants. Higher extracellular K⁺ elevations during spreading depolarizations in the mutants implicated a defect in extracellular K⁺ clearance. Altogether, these data reveal a mechanism of enhanced vulnerability to ischemic injury linked to abnormal extracellular ion homeostasis and susceptibility to ischemic depolarizations in CADASIL.     

Antipodean forensics: a comment on ANZFSS’s response to PCAST

Recently, one of the world’s leading scientific and technical advisory groups, the U.S. President’s Council of Advisors on Science and Technology (or PCAST), published an assessment of the research supporting several forensic science procedures in regular use. PCAST was particularly interested in pattern recognition or comparison procedures, specifically: simple and mixed DNA profiles; bite marks; latent fingerprints; firearms; footwear; and hair analysis. In its report and recommendations PCAST emphasised the primacy of validation. In response, the Council of the Australian and New Zealand Forensic Science Society produced a short statement suggesting that the PCAST report has limited application to forensic science in Australia and New Zealand. This short commentary offers an alternative perspective.     

The prognostic value of hypocholesterolemia in hospitalized patients

Clinical observations show that severe illness often leads to hypocholesterolemia. To verify this finding and to define the relationship between serum cholesterol and a patient's prognosis, a study was conducted in two large hospital populations. Of 24,000 and 61,463 adult patients (populations I and II) an average of 3.8% and 3.6% died in hospital, respectively. The mean serum cholesterol levels of patients who died was significantly lower than that of those who survived (163.6 mg/dl versus 217.8 mg/dl;P < 0.0001). The average cholesterol of surviving patients was similar to that of 6,543 healthy controls. During hospitalization serum cholesterol levels of 100 mg/dl were encountered in 1.2% and 3.6% of patients of populations I and II, respectively. The mortality of these hypocholesterolemic patients was about tenfold higher than average and showed a strong, inverse, linear relationship with serum cholesterol concentrations. Patients whose serum cholesterol level dropped to less than 45 mg/dl did not survive. These data show that in severely ill patients serum cholesterol may decline to very low concentrations, and the prognosis is reflected by the degree of hypocholesterolemia, which thus may serve as a clinically useful prognostic parameter. Correspondence to: E. Windler     

Prediction of function for the polyprenyl transferase subgroup in the isoprenoid synthase superfamily

The number of available protein sequences has increased exponentially with the advent of high-throughput genomic sequencing, creating a significant challenge for functional annotation. Here, we describe a large-scale study on assigning function to unknown members of the trans-polyprenyl transferase (E-PTS) subgroup in the isoprenoid synthase superfamily, which provides substrates for the biosynthesis of the more than 55,000 isoprenoid metabolites. Although the mechanism for determining the product chain length for these enzymes is known, there is no simple relationship between function and primary sequence, so that assigning function is challenging. We addressed this challenge through large-scale bioinformatics analysis of >5,000 putative polyprenyl transferases; experimental characterization of the chain-length specificity of 79 diverse members of this group; determination of 27 structures of 19 of these enzymes, including seven cocrystallized with substrate analogs or products; and the development and successful application of a computational approach to predict function that leverages available structural data through homology modeling and docking of possible products into the active site. The crystallographic structures and computational structural models of the enzyme–ligand complexes elucidate the structural basis of specificity. As a result of this study, the percentage of E-PTS sequences similar to functionally annotated ones (BLAST e-value ≤ 1e⁻⁷⁰) increased from 40.6 to 68.8%, and the percentage of sequences similar to available crystal structures increased from 28.9 to 47.4%. The high accuracy of our blind prediction of newly characterized enzymes indicates the potential to predict function to the complete polyprenyl transferase subgroup of the isoprenoid synthase superfamily computationally.The five-carbon molecules isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) are the fundamental building blocks for isoprenoid compounds. Beginning with DMAPP, a series of polyprenyl diphosphates with C₁₀ (geranyl diphosphate, GPP), C₁₅ (farnesyl diphosphate, FPP), C₂₀ (geranylgeranyl diphosphate, GGPP), C₂₅ (farnesylgeranyl diphosphate, FGPP), and higher molecular weight isoprenoid chains are synthesized by polyprenyl transferases (PTSs). With only a few exceptions, PTSs provide substrates for all but a few branch point enzymes for the biosynthesis of the more than 55,000 known isoprenoid metabolites, including monoterpenes, sesquiterpenes, diterpenes, sterols, carotenoids, ubiquinones, and prenylated proteins and peptides fulfilling essential roles in cells (Fig. S1) (1, 2).There are two distinct classes of PTSs, E-PTS forming trans bonds and Z-PTS forming cis bonds throughout chain elongation. The carbon skeletons of the great majority of isoprenoid metabolites are derived from products of E-PTSs, which share a common protein fold and two functionally important Asp-rich (DDXXD) motifs (1, 3, 4). E-PTSs synthesize linear allylic diphosphates ranging from C₁₀ to C₅₀, where functional assignment of these enzymes is linked to the chain length of their respective predominant products under nonforced conditions. As revealed by a variety of high-quality crystal structures, the enzymes typically are homodimers (5–7). In each monomer, the allylic binding region (S1) in the active site contains three Mg²⁺ ions ligated by two pairs of aspartates from both Asp-rich regions, which in turn holds the diphosphate of the allylic substrate in place, as illustrated with DMAPP in Fig. 1. The hydrocarbon tail of DMAPP extends into a pocket that accommodates the growing polyisoprenoid chain. The IPP binding region (S2) positions C4 of IPP near C1 of the allylic substrate. The isoprenoid moieties of the substrates are joined by a dissociative electrophilic alkylation initiated by cleavage of the carbon–oxygen bond of the allylic diphosphate to form a carbocation, which then attacks the C3–C4 double bond of allylic IPP to form a tertiary carbocationic intermediate that loses a proton from C2 to give the allylic product (8). The sequential addition of IPP to the growing chain proceeds through release of diphosphate from S1, rearrangement of the product from S2 to S1, and binding of another IPP in S2, followed by the same reaction as before (Fig. 1A). The polyisoprenoid chain grows into the elongation cavity flanked by helices D, F, G, and H of the protein near the dimer interface (Fig. 1 B and C) (7, 9). Following this elongation mechanism, these enzymes are named either according to their final product (e.g., farnesyl diphosphate synthase) or according to the longest ligand being transferred to the final product (in this case, geranyl transferase).Open in a separate windowFig. 1.Crystal structure of GGPP synthase (PDB ID 1RQI). (A) Active site S1 with DMAPP, Mg²⁺ ions, and Asp-rich motifs and active site S2 with IPP are highlighted. The electrophilic attack of the C₁ atom of DMAPP against the double bond of IPP after cleavage of diphosphate is indicated by the black arrow. (B and C) Side view (B) and top view (C) of the bioactive dimer with the active site and elongation cavity displayed. Helices D–H are identified by capital letters.Considerable effort has been made to investigate how chain elongation is terminated, establishing that steric hindrance of the growing chain in the elongation cavity is the main factor (1, 3, 9, 10). However, because the number of predicted sequences in the superfamily is so large, structural and enzymatic studies can be performed on only a small fraction of the sequences with likely E-PTS activity. We identified 5,839 such sequences at the initiation of this study in May 2011 (see below). At that time, only 46 individual sequences from 35 UniProt entries were functionally annotated based on published biochemical essays and thus available in the Gene Ontology Annotation (UniProt-GOA) database (11), and 61 had been structurally characterized with crystal structures available in the Protein Data Bank (PDB) database (12). Moreover, the rate of genome sequencing continues to increase exponentially, and even with on-going advances in high-throughput structural biology and in vitro screening methods, the gap between the number of known sequences and the number characterized experimentally will continue to grow. For this reason, reliable methods of inferring function of uncharacterized sequences are urgently needed. The highest-throughput and most widely applied approaches transfer functional annotations from characterized proteins to closely related proteins, because closely related proteins frequently are iso-functional. However, there is no simple sequence-based criterion that can be used universally to define when sequences are similar enough for the inference of iso-functionality to be made confidently (13). In the case of E-PTS enzymes, it has been demonstrated experimentally that chain-length specificity can be changed dramatically by a small number of mutations of key residues lining the elongation cavity (9, 14). Thus, in principle, even very closely related sequences could have different chain-length specificities. Furthermore, the previously characterized E-PTS enzymes were insufficient to allow inferences about the function of many members of this protein family, using any reasonable sequence-similarity cutoff, in part because many of the characterized enzymes were closely related to each other, leaving large regions of “sequence space” completely uncharacterized.We now describe a large-scale study that integrates available genomics data, in vitro experiments, X-ray crystallography, and computational approaches on a large set of representative members of the E-PTS subgroup to assign and, more importantly, to predict function for uncharacterized sequences. We show that computational tools can play a critical role in functional assignment. We use bioinformatics analysis and sequence clustering for target selection and place a particular emphasis on the use of structural information to make functional inferences. To this end, we determined 27 crystal structures and used these structures (in addition to available structures in the PDB) to create comparative models for 61 PTS enzymes for which structural information is lacking. Structures and models then were used to make predictions of chain-length specificity, using a ligand-docking method that evaluates the steric complementarity between various polyprenyl products and the elongation cavity. This structure-based approach to predicting function is validated through blind predictions on 74 PTS enzymes as well as on a subsequently obtained crystal structure in complex with a long-chain polyprenyl product.     

SOS1 is the second most common Noonan gene but plays no major role in cardio-facio-cutaneous syndrome

Background

Heterozygous gain‐of‐function mutations in various genes encoding proteins of the Ras‐MAPK signalling cascade have been identified as the genetic basis of Noonan syndrome (NS) and cardio‐facio‐cutaneous syndrome (CFCS). Mutations of SOS1, the gene encoding a guanine nucleotide exchange factor for Ras, have been the most recent discoveries in patients with NS, but this gene has not been studied in patients with CFCS.

Methods and results

We investigated SOS1 in a large cohort of patients with disorders of the NS–CFCS spectrum, who had previously tested negative for mutations in PTPN11, KRAS, BRAF, MEK1 and MEK2. Missense mutations of SOS1 were discovered in 28% of patients with NS. In contrast, none of the patients classified as having CFCS was found to carry a pathogenic sequence change in this gene.

Conclusion

We have confirmed SOS1 as the second major gene for NS. Patients carrying mutations in this gene have a distinctive phenotype with frequent ectodermal anomalies such as keratosis pilaris and curly hair. However, the clinical picture associated with SOS1 mutations is different from that of CFCS. These findings corroborate that, despite being caused by gain‐of‐function mutations in molecules belonging to the same pathway, NS and CFCS scarcely overlap genotypically.     

Glycemic control and type 1 diabetes: the differential impact of model of care and income

Hatherly K, Smith L, Overland J, Johnston C, Brown‐Singh L, Waller D, Taylor S. Glycemic control and type 1 diabetes: the differential impact of model of care and income. Objective: To examine the effect of model of care (specialist care vs. shared care), and income, on glycemic control in a sample of young people with type 1 diabetes. Methods: A total of 158 children and young people with type 1 diabetes, aged 8–19 yr, and their families, were recruited independent of their source of care as part of a longitudinal, cross‐sectional exploratory study. At enrollment, participants completed a series of questionnaires and underwent a structured interview to gather data regarding the type of specialist and healthcare services attended, as well as demographic, healthcare, and self‐care information. Capillary sample was taken for HbA1c determination. Results: The mean HbA1c for the group as a whole was 8.6 ± 1.4%. There was no effect for model of care on glycemic control. However, young people living in households with a family income of less than AUS$83 000 (US$73 500) per year had a significantly higher mean HbA1c than their counterparts reporting a higher household income (8.8 ± 1.4% vs. 8.3 ± 1.1%; p = 0.019). Conclusion: Although no differences were found with respect to the short‐term impact of specialist vs. shared care, it is evident that more support is required to improve glycemic control in this sample of young people where the mean level of HbA1c was significantly higher than target. Further research is also indicated to determine the relationship between glycemic control and socioeconomic status.