Modelling amorphous computations with transcription networks

The power of electronic computation is due in part to the development of modular gate structures that can be coupled to carry out sophisticated logical operations and whose performance can be readily modelled. However, the equivalences between electronic and biochemical operations are far from obvious. In order to help cross between these disciplines, we develop an analogy between complementary metal oxide semiconductor and transcriptional logic gates. We surmise that these transcriptional logic gates might prove to be useful in amorphous computations and model the abilities of immobilized gates to form patterns. Finally, to begin to implement these computations, we design unique hairpin transcriptional gates and then characterize these gates in a binary latch similar to that already demonstrated by Kim et al. (Kim, White & Winfree 2006 Mol. Syst. Biol. 2, 68 (doi:10.1038/msb4100099)). The hairpin transcriptional gates are uniquely suited to the design of a complementary NAND gate that can serve as an underlying basis of molecular computing that can output matter rather than electronic information

Redox properties of human hemoglobin in complex with fractionated dimeric and polymeric human haptoglobin

Haptoglobin (Hp) is an abundant and conserved plasma glycoprotein, which binds acellular adult hemoglobin (Hb) dimers with high affinity and facilitates their rapid clearance from circulation after hemolysis. Humans possess three main phenotypes of Hp, designated Hp 1-1, Hp 2-1, and Hp 2-2. These variants exhibit diverse structural configurations and have been reported to be functionally nonequivalent. We have investigated the functional and redox properties of Hb–Hp complexes prepared using commercially fractionated Hp and found that all forms exhibit similar behavior. The rate of Hb dimer binding to Hp occurs with bimolecular rate constants of ~0.9 μM−1 s−1, irrespective of the type of Hp assayed. Although Hp binding does accelerate the observed rate of HbO2 autoxidation by dissociating Hb tetramers into dimers, the rate observed for these bound dimers is three- to fourfold slower than that of Hb dimers free in solution. Co-incubation of ferric Hb with any form of Hp inhibits heme loss to below detectable levels. Intrinsic redox potentials (E1/2) of the ferric/ferrous pair of each Hb–Hp complex are similar, varying from +54 to +59 mV (vs NHE), and are essentially the same as reported by us previously for Hb–Hp complexes prepared from unfractionated Hp. All Hb–Hp complexes generate similar high amounts of ferryl Hb after exposure to hydrogen peroxide. Electron paramagnetic resonance data indicate that the yields of protein-based radicals during this process are approximately 4 to 5% and are unaffected by the variant of Hp assayed. These data indicate that the Hp fractions examined are equivalent to one another with respect to Hb binding and associated stability and redox properties and that this result should be taken into account in the design of phenotype-specific Hp therapeutics aimed at countering Hb-mediated vascular disease

A crotonyl-CoA reductase-carboxylase independent pathway for assembly of unusual alkylmalonyl-CoA polyketide synthase extender units

Type I modular polyketide synthases assemble diverse bioactive natural products. Such multienzymes typically use malonyl and methylmalonyl-CoA building blocks for polyketide chain assembly. However, in several cases more exotic alkylmalonyl-CoA extender units are also known to be incorporated. In all examples studied to date, such unusual extender units are biosynthesized via reductive carboxylation of α, β-unsaturated thioesters catalysed by crotonyl-CoA reductase/carboxylase (CCRC) homologues. Here we show using a chemically-synthesized deuterium-labelled mechanistic probe, and heterologous gene expression experiments that the unusual alkylmalonyl-CoA extender units incorporated into the stambomycin family of polyketide antibiotics are assembled by direct carboxylation of medium chain acyl-CoA thioesters. X-ray crystal structures of the unusual β-subunit of the acyl-CoA carboxylase (YCC) responsible for this reaction, alone and in complex with hexanoyl-CoA, reveal the molecular basis for substrate recognition, inspiring the development of methodology for polyketide bio-orthogonal tagging via incorporation of 6-azidohexanoic acid and 8-nonynoic acid into novel stambomycin analogues

Structural basis for chain release from the enacyloxin polyketide synthase

Modular polyketide synthases and nonribosomal peptide synthetases are molecular assembly lines consisting of several multienzyme subunits that undergo dynamic self-assembly to form a functional mega-complex. N- and C-terminal docking domains are usually responsible for mediating interactions between subunits. Here we show that communication between two nonribosomal peptide synthetase subunits responsible for chain release from the enacyloxin polyketide synthase, which assembles an antibiotic with promising activity against Acinetobacter baumannii, is mediated by an intrinsically disordered short linear motif and a ß-hairpin docking domain. The structures, interactions and dynamics of these subunits are characterised using several complementary biophysical techniques, providing extensive insights into binding and catalysis. Bioinformatics analyses reveal that short linear motif/ß-hairpin docking domain pairs mediate subunit interactions in numerous nonribosomal peptide and hybrid polyketide-nonribosomal peptide synthetases, including those responsible for assembling several important drugs. Short linear motifs and ß-hairpin docking domains from heterologous systems are shown to interact productively, highlighting the potential of such interfaces as tools for biosynthetic engineering

Publisher Correction: Deep coverage whole genome sequences and plasma lipoprotein(a) in individuals of European and African ancestries.

The original version of this article contained an error in the name of the author Ramachandran S. Vasan, which was incorrectly given as Vasan S. Ramachandran. This has now been corrected in both the PDF and HTML versions of the article

Deep coverage whole genome sequences and plasma lipoprotein(a) in individuals of European and African ancestries.

Lipoprotein(a), Lp(a), is a modified low-density lipoprotein particle that contains apolipoprotein(a), encoded by LPA, and is a highly heritable, causal risk factor for cardiovascular diseases that varies in concentrations across ancestries. Here, we use deep-coverage whole genome sequencing in 8392 individuals of European and African ancestry to discover and interpret both single-nucleotide variants and copy number (CN) variation associated with Lp(a). We observe that genetic determinants between Europeans and Africans have several unique determinants. The common variant rs12740374 associated with Lp(a) cholesterol is an eQTL for SORT1 and independent of LDL cholesterol. Observed associations of aggregates of rare non-coding variants are largely explained by LPA structural variation, namely the LPA kringle IV 2 (KIV2)-CN. Finally, we find that LPA risk genotypes confer greater relative risk for incident atherosclerotic cardiovascular diseases compared to directly measured Lp(a), and are significantly associated with measures of subclinical atherosclerosis in African Americans

Anti-Nogo-A Immunotherapy Does Not Alter Hippocampal Neurogenesis after Stroke in Adult Rats

Ischemic stroke is a leading cause of adult disability, including cognitive impairment. Our laboratory has previously shown that treatment with function-blocking antibodies against the neurite growth inhibitory protein Nogo-A promotes functional recovery after stroke in adult and aged rats, including enhancing spatial memory performance, for which the hippocampus is critically important. Since spatial memory has been linked to hippocampal neurogenesis, we investigated whether anti-Nogo-A treatment increases hippocampal neurogenesis after stroke. Adult rats were subject to permanent middle cerebral artery occlusion followed 1 week later by 2 weeks of antibody treatment. Cellular proliferation in the dentate gyrus was quantified at the end of treatment, and the number of newborn neurons was determined at 8 weeks post-stroke. Treatment with both anti-Nogo-A and control antibodies stimulated the accumulation of new microglia/macrophages in the dentate granule cell layer, but neither treatment increased cellular proliferation or the number of newborn neurons above stroke-only levels. These results suggest that anti-Nogo-A immunotherapy does not increase post-stroke hippocampal neurogenesis

A Multi-Parameter, High-Content, High-Throughput Screening Platform to Identify Natural Compounds that Modulate Insulin and Pdx1 Expression

Diabetes is a devastating disease that is ultimately caused by the malfunction or loss of insulin-producing pancreatic beta-cells. Drugs capable of inducing the development of new beta-cells or improving the function or survival of existing beta-cells could conceivably cure this disease. We report a novel high-throughput screening platform that exploits multi-parameter high-content analysis to determine the effect of compounds on beta-cell survival, as well as the promoter activity of two key beta-cell genes, insulin and pdx1. Dispersed human pancreatic islets and MIN6 beta-cells were infected with a dual reporter lentivirus containing both eGFP driven by the insulin promoter and mRFP driven by the pdx1 promoter. B-score statistical transformation was used to correct systemic row and column biases. Using this approach and 5 replicate screens, we identified 7 extracts that reproducibly changed insulin and/or pdx1 promoter activity from a library of 1319 marine invertebrate extracts. The ability of compounds purified from these extracts to significantly modulate insulin mRNA levels was confirmed with real-time PCR. Insulin secretion was analyzed by RIA. Follow-up studies focused on two lead compounds, one that stimulates insulin gene expression and one that inhibits insulin gene expression. Thus, we demonstrate that multi-parameter, high-content screening can identify novel regulators of beta-cell gene expression, such as bivittoside D. This work represents an important step towards the development of drugs to increase insulin expression in diabetes and during in vitro differentiation of beta-cell replacements

Objective sequence-based subfamily classifications of mouse homeodomains reflect their in vitro DNA-binding preferences

Classifying proteins into subgroups with similar molecular function on the basis of sequence is an important step in deriving reliable functional annotations computationally. So far, however, available classification procedures have been evaluated against protein subgroups that are defined by experts using mainly qualitative descriptions of molecular function. Recently, in vitro DNA-binding preferences to all possible 8-nt DNA sequences have been measured for 178 mouse homeodomains using protein-binding microarrays, offering the unprecedented opportunity of evaluating the classification methods against quantitative measures of molecular function. To this end, we automatically derive homeodomain subtypes from the DNA-binding data and independently group the same domains using sequence information alone. We test five sequence-based methods, which use different sequence-similarity measures and algorithms to group sequences. Results show that methods that optimize the classification robustness reflect well the detailed functional specificity revealed by the experimental data. In some of these classifications, 73–83% of the subfamilies exactly correspond to, or are completely contained in, the function-based subtypes. Our findings demonstrate that certain sequence-based classifications are capable of yielding very specific molecular function annotations. The availability of quantitative descriptions of molecular function, such as DNA-binding data, will be a key factor in exploiting this potential in the future.Canadian Institutes of Health Research (MOP#82940)Sickkids FoundationOntario Research FundNational Science Foundation (U.S.)National Human Genome Research Institute (U.S.) (R01 HG003985

Readmissions after general surgery: a prospective multicenter audit

Background: Readmission rates after surgical procedures are viewed as a marker of quality of care and as a driver to improve outcomes in the United Kingdom, they are not remunerated. However, readmissions are not wholly avoidable. The aim of this study was to develop a regional overview of readmissions to determine the proportion that might be avoidable and to examine predictors of readmissions at a unit level. Methods: We undertook a prospective multicenter audit of readmissions following National Health Service funded general surgical procedures in five National Health Service hospitals and three independent sector providers over a 2-wk period. Basic demographic and procedure data were captured. Readmissions to hospitals were identified through acute admissions lists. Reason for readmission was identified, and the readmission data assessed by a senior surgical doctor as to whether it was avoidable. Results: We identified 752 operations in the study period with all followed up to 30 d. The overall rate of readmissions was 4.7%, with 40% of these judged as being potentially avoidable. Pain and wound problems accounted for the vast majority of avoidable readmissions. The number of unavoidable readmissions was correlated with the workload of each center (r ¼ 0.63, P ¼ 0.06) and as with the higher (British United Provident Association) complexity of surgery (r ¼ 0.90, P ¼ 0.01). Patient and demographic factors were not associated with readmissions. Conclusions: This prospective audit describes readmission rates after general surgery. Volume and complexity of work are associated with readmission rates. A large proportion of readmissions could be reduced by attention to analgesia and outpatient arrangements for wound management