Protein engineering enables a soakable crystal form of human CDK7 primed for high-throughput crystallography and structure-based drug design.

Cyclin dependent kinase 7 (CDK7) is an important therapeutic kinase best known for its dual role in cell cycle regulation and gene transcription. Here, we describe the application of protein engineering to generate constructs leading to high resolution crystal structures of human CDK7 in both active and inactive conformations. The active state of the kinase was crystallized by incorporation of an additional surface residue mutation (W132R) onto the double phosphomimetic mutant background (S164D and T170E) that yielded the inactive kinase structure. A novel back-soaking approach was developed to determine crystal structures of several clinical and pre-clinical inhibitors of this kinase, demonstrating the potential utility of the crystal system for structure-based drug design (SBDD). The crystal structures help to rationalize the mode of inhibition and the ligand selectivity profiles versus key anti-targets. The protein engineering approach described here illustrates a generally applicable strategy for structural enablement of challenging molecular targets.

Tunable Release of Ions from Graphene Oxide Laminates for Sustained Antibacterial Activity in a Biomimetic Environment.

Silver has long been recognized for its potent antimicrobial properties, but achieving a slow and longer-term delivery of silver ions presents significant challenges. Previous efforts to control silver ion dosages have struggled to sustain release for extended periods in biomimetic environments, especially in the presence of complex proteins. This challenge is underscored by the absence of technology for sustaining antimicrobial activity, especially in the context of orthopedic implants where long-term efficacy, extending beyond 7 days, is essential. In this study, the tunable, slow, and longer-term release of silver ions from the two-dimensional (2D) nanocapillaries of graphene oxide (GO) laminates incorporated with silver ions (Ag-GO) for antimicrobial applications are successfully demonstrated. To closely mimic a physiologically relevant serum-based environment, a novel in vitro study model using 100% fetal bovine serum (FBS) is introduced as the test medium for microbiology, biocompatibility, and bioactivity studies. To emulate fluid circulation in a physiological environment, the in vitro studies are challenged with serum exchange protocols on different days. The findings show that the Ag-GO coating can sustainably release silver ions at a minimum dosage of 10 µg cm-2 day-1, providing an effective and sustained antimicrobial barrier for over ten days.

Fragment-Based Discovery of Allosteric Inhibitors of SH2 Domain-Containing Protein Tyrosine Phosphatase-2 (SHP2).

The ubiquitously expressed protein tyrosine phosphatase SHP2 is required for signaling downstream of receptor tyrosine kinases (RTKs) and plays a role in regulating many cellular processes. Genetic knockdown and pharmacological inhibition of SHP2 suppresses RAS/MAPK signaling and inhibit the proliferation of RTK-driven cancer cell lines. Here, we describe the first reported fragment-to-lead campaign against SHP2, where X-ray crystallography and biophysical techniques were used to identify fragments binding to multiple sites on SHP2. Structure-guided optimization, including several computational methods, led to the discovery of two structurally distinct series of SHP2 inhibitors binding to the previously reported allosteric tunnel binding site (Tunnel Site). One of these series was advanced to a low-nanomolar lead that inhibited tumor growth when dosed orally to mice bearing HCC827 xenografts. Furthermore, a third series of SHP2 inhibitors was discovered binding to a previously unreported site, lying at the interface of the C-terminal SH2 and catalytic domains.

Cell Surface Transporters and Novel Drug Developments.

Despite the numerous scientific and technological advances made within the last decade the attrition rates for new drug discovery remain as high as 95% for anticancer drugs. Recent drug development has been in part guided by Lipinski's Rule of 5 (Ro5) even though many approved drugs do not comply to these rules. With Covid-19 vaccine development strategy dramatically accelerating drug development perhaps it is timely to question the generic drug development process itself to find a more efficient, cost effective, and successful approach. It is widely believed that drugs permeate cells via two methods: phospholipid bilayer diffusion and carrier mediated transporters. However, emerging evidence suggests that carrier mediated transport may be the primary mechanism of drug uptake and not diffusion as long believed. Computational biology increasingly assists drug design to achieve desirable absorption, distribution, metabolism, elimination and toxicity (ADMET) properties. Perfecting drug entry into target cells as a prerequisite to intracellular drug action is a logical and compelling route and is expected to reduce drug attrition rates, particularly gaining favour amongst chronic lifelong therapeutics. Novel drug development is rapidly expanding from the utilisation of beyond the rule of five (bRo5) to pulsatile drug delivery systems and fragment based drug design. Utilising transporters as drug targets and advocating bRo5 molecules may be the solution to increasing drug specificity, reducing dosage and toxicity and thus revolutionising drug development. This review explores the development of cell surface transporter exploitation in drug development and the relationship with improved therapeutic index.

An Analysis of Mechanisms for Cellular Uptake of miRNAs to Enhance Drug Delivery and Efficacy in Cancer Chemoresistance.

Up until recently, it was believed that pharmaceutical drugs and their metabolites enter into the cell to gain access to their targets via simple diffusion across the hydrophobic lipid cellular membrane, at a rate which is based on their lipophilicity. An increasing amount of evidence indicates that the phospholipid bilayer-mediated drug diffusion is in fact negligible, and that drugs pass through cell membranes via proteinaceous membrane transporters or carriers which are normally used for the transportation of nutrients and intermediate metabolites. Drugs can be targeted to specific cells and tissues which express the relevant transporters, leading to the design of safe and efficacious treatments. Furthermore, transporter expression levels can be manipulated, systematically and in a high-throughput manner, allowing for considerable progress in determining which transporters are used by specific drugs. The ever-expanding field of miRNA therapeutics is not without its challenges, with the most notable one being the safe and effective delivery of the miRNA mimic/antagonist safely to the target cell cytoplasm for attaining the desired clinical outcome, particularly in miRNA-based cancer therapeutics, due to the poor efficiency of neo-vascular systems revolting around the tumour site, brought about by tumour-induced angiogenesis. This acquisition of resistance to several types of anticancer drugs can be as a result of an upregulation of efflux transporters expression, which eject drugs from cells, hence lowering drug efficacy, resulting in multidrug resistance. In this article, the latest available data on human microRNAs has been reviewed, together with the most recently described mechanisms for miRNA uptake in cells, for future therapeutic enhancements against cancer chemoresistance.

Viral Involvement in Alzheimer's Disease.

Alzheimer's disease (AD) is characterized by the presence of ß-amyloid plaques (Aß) and neurofibrillary tangles (NFTs) in the brain. The prevalence of the disease is increasing and is expected to reach 141 million cases by 2050. Despite the risk factors associated with the disease, there is no known causative agent for AD. Clinical trials with many drugs have failed over the years, and no therapeutic has been approved for AD. There is increasing evidence that pathogens are found in the brains of AD patients and controls, such as human herpes simplex virus-1 (HSV-1). Given the lack of a human model, the route for pathogen entry into the brain remains open for scrutiny and may include entry via a disturbed blood-brain barrier or the olfactory nasal route. Many factors can contribute to the pathogenicity of HSV-1, such as the ability of HSV-1 to remain latent, tau protein phosphorylation, increased accumulation of Aß invivo and in vitro, and repeated cycle of reactivation if immunocompromised. Intriguingly, valacyclovir, a widely used drug for the treatment of HSV-1 and HSV-2 infection, has shown patient improvement in cognition compared to controls in AD clinical studies. We discuss the potential role of HSV-1 in AD pathogenesis and argue for further studies to investigate this relationship.

An untargeted metabolomics strategy to measure differences in metabolite uptake and excretion by mammalian cell lines.

INTRODUCTION: It is widely but erroneously believed that drugs get into cells by passing through the phospholipid bilayer portion of the plasma and other membranes. Much evidence shows, however, that this is not the case, and that drugs cross biomembranes by hitchhiking on transporters for other natural molecules to which these drugs are structurally similar. Untargeted metabolomics can provide a method for determining the differential uptake of such metabolites. OBJECTIVES: Blood serum contains many thousands of molecules and provides a convenient source of biologically relevant metabolites. Our objective was to detect and identify metabolites present in serum, but to also establish a method capable of measure their uptake and secretion by different cell lines. METHODS: We develop an untargeted LC-MS/MS method to detect a broad range of compounds present in human serum. We apply this to the analysis of the time course of the uptake and secretion of metabolites in serum by several human cell lines, by analysing changes in the serum that represents the extracellular phase (the 'exometabolome' or metabolic footprint). RESULTS: Our method measures some 4000-5000 metabolic features in both positive and negative electrospray ionisation modes. We show that the metabolic footprints of different cell lines differ greatly from each other. CONCLUSION: Our new, 15-min untargeted metabolome method allows for the robust and convenient measurement of differences in the uptake of serum compounds by cell lines following incubation in serum. This will enable future research to study these differences in multiple cell lines that will relate this to transporter expression, thereby advancing our knowledge of transporter substrates, both natural and xenobiotic compounds.

MicroRNA and mRNA profiling in the idiopathic inflammatory myopathies.

BACKGROUND: The idiopathic inflammatory myopathies (IIMs) are heterogeneous autoimmune conditions of skeletal muscle inflammation and weakness. MicroRNAs (miRNAs) are short, non-coding RNA which regulate gene expression of target mRNAs. The aim of this study was to profile miRNA and mRNA in IIM and identify miRNA-mRNA relationships which may be relevant to disease. METHODS: mRNA and miRNA in whole blood samples from 7 polymyositis (PM), 7 dermatomyositis (DM), 5 inclusion body myositis and 5 non-myositis controls was profiled using next generation RNA sequencing. Gene ontology and pathway analyses were performed using GOseq and Ingenuity Pathway Analysis. Dysregulation of miRNAs and opposite dysregulation of predicted target mRNAs in IIM subgroups was validated using RTqPCR and investigated by transfecting human skeletal muscle cells with miRNA mimic. RESULTS: Analysis of differentially expressed genes showed that interferon signalling, and anti-viral response pathways were upregulated in PM and DM compared to controls. An anti-Jo1 autoantibody positive subset of PM and DM (n = 5) had more significant upregulation and predicted activation of interferon signalling and highlighted T-helper (Th1 and Th2) cell pathways. In miRNA profiling miR-96-5p was significantly upregulated in PM, DM and the anti-Jo1 positive subset. RTqPCR replicated miR-96-5p upregulation and predicted mRNA target (ADK, CD28 and SLC4A10) downregulation. Transfection of a human skeletal muscle cell line with miR-96-5p mimic resulted in significant downregulation of ADK. CONCLUSION: MiRNA and mRNA profiling identified dysregulation of interferon signalling, anti-viral response and T-helper cell pathways, and indicates a possible role for miR-96-5p regulation of ADK in pathogenesis of IIM.

The role and robustness of the Gini coefficient as an unbiased tool for the selection of Gini genes for normalising expression profiling data.

We recently introduced the Gini coefficient (GC) for assessing the expression variation of a particular gene in a dataset, as a means of selecting improved reference genes over the cohort ('housekeeping genes') typically used for normalisation in expression profiling studies. Those genes (transcripts) that we determined to be useable as reference genes differed greatly from previous suggestions based on hypothesis-driven approaches. A limitation of this initial study is that a single (albeit large) dataset was employed for both tissues and cell lines. We here extend this analysis to encompass seven other large datasets. Although their absolute values differ a little, the Gini values and median expression levels of the various genes are well correlated with each other between the various cell line datasets, implying that our original choice of the more ubiquitously expressed low-Gini-coefficient genes was indeed sound. In tissues, the Gini values and median expression levels of genes showed a greater variation, with the GC of genes changing with the number and types of tissues in the data sets. In all data sets, regardless of whether this was derived from tissues or cell lines, we also show that the GC is a robust measure of gene expression stability. Using the GC as a measure of expression stability we illustrate its utility to find tissue- and cell line-optimised housekeeping genes without any prior bias, that again include only a small number of previously reported housekeeping genes. We also independently confirmed this experimentally using RT-qPCR with 40 candidate GC genes in a panel of 10 cell lines. These were termed the Gini Genes. In many cases, the variation in the expression levels of classical reference genes is really quite huge (e.g. 44 fold for GAPDH in one data set), suggesting that the cure (of using them as normalising genes) may in some cases be worse than the disease (of not doing so). We recommend the present data-driven approach for the selection of reference genes by using the easy-to-calculate and robust GC.

Involvement of multiple influx and efflux transporters in the accumulation of cationic fluorescent dyes by Escherichia coli.

BACKGROUND: It is widely believed that most xenobiotics cross biomembranes by diffusing through the phospholipid bilayer, and that the use of protein transporters is an occasional adjunct. According to an alternative view, phospholipid bilayer transport is negligible, and several different transporters may be involved in the uptake of an individual molecular type. We recognise here that the availability of gene knockout collections allows one to assess the contributions of all potential transporters, and flow cytometry based on fluorescence provides a convenient high-throughput assay for xenobiotic uptake in individual cells. RESULTS: We used high-throughput flow cytometry to assess the ability of individual gene knockout strains of E coli to take up two membrane-permeable, cationic fluorescent dyes, namely the carbocyanine diS-C3(5) and the DNA dye SYBR Green. Individual strains showed a large range of distributions of uptake. The range of modal steady-state uptakes for the carbocyanine between the different strains was 36-fold. Knockouts of the ATP synthase α- and ß-subunits greatly inhibited uptake, implying that most uptake was ATP-driven rather than being driven by a membrane potential. Dozens of transporters changed the steady-state uptake of the dye by more than 50% with respect to that of the wild type, in either direction (increased or decreased); knockouts of known influx and efflux transporters behaved as expected, giving credence to the general strategy. Many of the knockouts with the most reduced uptake were transporter genes of unknown function ('y-genes'). Similarly, several overexpression variants in the 'ASKA' collection had the anticipated, opposite effects. Similar results were obtained with SYBR Green (the range being approximately 69-fold). Although it too contains a benzothiazole motif there was negligible correlation between its uptake and that of the carbocyanine when compared across the various strains (although the membrane potential is presumably the same in each case). CONCLUSIONS: Overall, we conclude that the uptake of these dyes may be catalysed by a great many transporters of putatively broad and presently unknown specificity, and that the very large range between the 'lowest' and the 'highest' levels of uptake, even in knockouts of just single genes, implies strongly that phospholipid bilayer transport is indeed negligible. This work also casts serious doubt upon the use of such dyes as quantitative stains for representing either bioenergetic parameters or the amount of cellular DNA in unfixed cells (in vivo). By contrast, it opens up their potential use as transporter assay substrates in high-throughput screening.

A brain-permeable inhibitor of the neurodegenerative disease target kynurenine 3-monooxygenase prevents accumulation of neurotoxic metabolites.

Dysregulation of the kynurenine pathway (KP) leads to imbalances in neuroactive metabolites associated with the pathogenesis of several neurodegenerative disorders, including Huntington's disease (HD). Inhibition of the enzyme kynurenine 3-monooxygenase (KMO) in the KP normalises these metabolic imbalances and ameliorates neurodegeneration and related phenotypes in several neurodegenerative disease models. KMO is thus a promising candidate drug target for these disorders, but known inhibitors are not brain permeable. Here, 19 new KMO inhibitors have been identified. One of these (1) is neuroprotective in a Drosophila HD model but is minimally brain penetrant in mice. The prodrug variant (1b) crosses the blood-brain barrier, releases 1 in the brain, thereby lowering levels of 3-hydroxykynurenine, a toxic KP metabolite linked to neurodegeneration. Prodrug 1b will advance development of targeted therapies against multiple neurodegenerative and neuroinflammatory diseases in which KP likely plays a role, including HD, Alzheimer's disease, and Parkinson's disease.

Very rapid flow cytometric assessment of antimicrobial susceptibility during the apparent lag phase of microbial (re)growth.

Rapid changes in the number and flow cytometric behaviour of cells of E. coli taken from a stationary phase and inoculated into rich medium.Cells of E. coli were grown in LB medium, taken from a stationary phase of 2-4 h, and re-inoculated into fresh media at a concentration (105 ml-1 or lower) characteristic of bacteriuria. Flow cytometry was used to assess how quickly we could detect changes in cell size, number, membrane energization (using a carbocyanine dye) and DNA distribution. It transpired that while the lag phase observable macroscopically via bulk OD measurements could be as long as 4 h, the true lag phase could be less than 15-20 min, and was accompanied by many observable biochemical changes. Antibiotics to which the cells were sensitive affected these changes within 20 min of re-inoculation, providing the possibility of a very rapid antibiotic susceptibility test on a timescale compatible with a visit to a GP clinic. The strategy was applied successfully to genuine potential urinary tract infection (UTI) samples taken from a doctor's surgery. The methods developed could prove of considerable value in ensuring the correct prescription and thereby lowering the spread of antimicrobial resistance.

GeneGini: Assessment via the Gini Coefficient of Reference "Housekeeping" Genes and Diverse Human Transporter Expression Profiles.

The expression levels of SLC or ABC membrane transporter transcripts typically differ 100- to 10,000-fold between different tissues. The Gini coefficient characterizes such inequalities and here is used to describe the distribution of the expression of each transporter among different human tissues and cell lines. Many transporters exhibit extremely high Gini coefficients even for common substrates, indicating considerable specialization consistent with divergent evolution. The expression profiles of SLC transporters in different cell lines behave similarly, although Gini coefficients for ABC transporters tend to be larger in cell lines than in tissues, implying selection. Transporter genes are significantly more heterogeneously expressed than the members of most non-transporter gene classes. Transcripts with the stablest expression have a low Gini index and often differ significantly from the "housekeeping" genes commonly used for normalization in transcriptomics/qPCR studies. PCBP1 has a low Gini coefficient, is reasonably expressed, and is an excellent novel reference gene. The approach, referred to as GeneGini, provides rapid and simple characterization of expression-profile distributions and improved normalization of genome-wide expression-profiling data.

Integrative analysis of rewired central metabolism in temozolomide resistant cells.

An authenticated U87MG clonal glioblastoma cell line was investigated to identify a sub-population of neurospheroidal (NSP) cells within the main epithelial population (U87MG). The NSP cells sorted using Fluorescence Assisted Cell Sorting (FACS) showed varied morphology, 30% lower growth rates, 40% higher IC50 values for temozolomide drug and could differentiate into the glial cell type (NDx). Metabolite profiling using HR-LCMS identified glucose, glutamine and serine in both populations and tryptophan only in U87MG as growth limiting substrates. Glycine, alanine, glutamate and proline were secreted by U87MG, however proline and glycine were re-utilized in NSP. Exo-metabolite profiling and phenotypic microarrays identified differential metabolism of primary carbon sources glucose and derived pyruvate for U87MG; glutamine and derived glutamate metabolism in NSP. Differential mRNA abundance of AKT1, PTEN, PIK3CA controlling metabolism, drug efflux, nutrient transport and epigenetic control MDM2 are potentially critical in shaping DNA methylation effects of temozolomide. Our study provides a new insight into the combined effect of these factors leading to temozolomide resistance in NSP.

Endoplasmic reticulum chaperone prolyl 4-hydroxylase, beta polypeptide (P4HB) promotes malignant phenotypes in glioma via MAPK signaling.

Endoplasmic reticulum (ER) chaperone Prolyl 4-hydroxylase, beta polypeptide (P4HB) has previously been identified as a novel target for chemoresistance in glioblastoma multiforme (GBM). Yet its functional roles in glioma carcinogenesis remain elusive. In clinical analysis using human glioma specimens and Gene Expression Omnibus (GEO) profiles, we found that aberrant expression of P4HB was correlated with high-grade malignancy and an angiogenic phenotype in glioma. Furthermore, P4HB upregulation conferred malignant characteristics including proliferation, invasion, migration and angiogenesis in vitro, and increased tumor growth in vivo via the mitogen-activated protein kinase (MAPK) signaling pathway. Pathway analysis suggested genetic and pharmacologic inhibition of P4HB suppressed MAPK expression and its downstream targets were involved in angiogenesis and invasion. This is the first study that demonstrates the oncogenic roles of P4HB and its underlying mechanism in glioma. Since tumor invasion and Vascularisation are typical hallmarks in malignant glioma, our findings uncover a promising anti-glioma mechanism through P4HB-mediated retardation of MAPK signal transduction.

Enhancing Drug Efficacy and Therapeutic Index through Cheminformatics-Based Selection of Small Molecule Binary Weapons That Improve Transporter-Mediated Targeting: A Cytotoxicity System Based on Gemcitabine.

The transport of drug molecules is mainly determined by the distribution of influx and efflux transporters for which they are substrates. To enable tissue targeting, we sought to develop the idea that we might affect the transporter-mediated disposition of small-molecule drugs via the addition of a second small molecule that of itself had no inhibitory pharmacological effect but that influenced the expression of transporters for the primary drug. We refer to this as a "binary weapon" strategy. The experimental system tested the ability of a molecule that on its own had no cytotoxic effect to increase the toxicity of the nucleoside analog gemcitabine to Panc1 pancreatic cancer cells. An initial phenotypic screen of a 500-member polar drug (fragment) library yielded three "hits." The structures of 20 of the other 2,000 members of this library suite had a Tanimoto similarity greater than 0.7 to those of the initial hits, and each was itself a hit (the cheminformatics thus providing for a massive enrichment). We chose the top six representatives for further study. They fell into three clusters whose members bore reasonable structural similarities to each other (two were in fact isomers), lending strength to the self-consistency of both our conceptual and experimental strategies. Existing literature had suggested that indole-3-carbinol might play a similar role to that of our fragments, but in our hands it was without effect; nor was it structurally similar to any of our hits. As there was no evidence that the fragments could affect toxicity directly, we looked for effects on transporter transcript levels. In our hands, only the ENT1-3 uptake and ABCC2,3,4,5, and 10 efflux transporters displayed measurable transcripts in Panc1 cultures, along with a ribonucleoside reductase RRM1 known to affect gemcitabine toxicity. Very strikingly, the addition of gemcitabine alone increased the expression of the transcript for ABCC2 (MRP2) by more than 12-fold, and that of RRM1 by more than fourfold, and each of the fragment "hits" served to reverse this. However, an inhibitor of ABCC2 was without significant effect, implying that RRM1 was possibly the more significant player. These effects were somewhat selective for Panc cells. It seems, therefore, that while the effects we measured were here mediated more by efflux than influx transporters, and potentially by other means, the binary weapon idea is hereby fully confirmed: it is indeed possible to find molecules that manipulate the expression of transporters that are involved in the bioactivity of a pharmaceutical drug. This opens up an entirely new area, that of chemical genomics-based drug targeting.

Computing exponentially faster: implementing a non-deterministic universal Turing machine using DNA.

The theory of computer science is based around universal Turing machines (UTMs): abstract machines able to execute all possible algorithms. Modern digital computers are physical embodiments of classical UTMs. For the most important class of problem in computer science, non-deterministic polynomial complete problems, non-deterministic UTMs (NUTMs) are theoretically exponentially faster than both classical UTMs and quantum mechanical UTMs (QUTMs). However, no attempt has previously been made to build an NUTM, and their construction has been regarded as impossible. Here, we demonstrate the first physical design of an NUTM. This design is based on Thue string rewriting systems, and thereby avoids the limitations of most previous DNA computing schemes: all the computation is local (simple edits to strings) so there is no need for communication, and there is no need to order operations. The design exploits DNA's ability to replicate to execute an exponential number of computational paths in P time. Each Thue rewriting step is embodied in a DNA edit implemented using a novel combination of polymerase chain reactions and site-directed mutagenesis. We demonstrate that the design works using both computational modelling and in vitro molecular biology experimentation: the design is thermodynamically favourable, microprogramming can be used to encode arbitrary Thue rules, all classes of Thue rule can be implemented, and non-deterministic rule implementation. In an NUTM, the resource limitation is space, which contrasts with classical UTMs and QUTMs where it is time. This fundamental difference enables an NUTM to trade space for time, which is significant for both theoretical computer science and physics. It is also of practical importance, for to quote Richard Feynman 'there's plenty of room at the bottom'. This means that a desktop DNA NUTM could potentially utilize more processors than all the electronic computers in the world combined, and thereby outperform the world's current fastest supercomputer, while consuming a tiny fraction of its energy.

Peripheral blood RNA gene expression in children with pneumococcal meningitis: a prospective case-control study.

INTRODUCTION: Invasive pneumococcal disease (IPD), caused by Streptococcus pneumoniae, is a leading cause of pneumonia, meningitis and septicaemia worldwide, with increased morbidity and mortality in HIV-infected children. OBJECTIVES: We aimed to compare peripheral blood expression profiles between HIV-infected and uninfected children with pneumococcal meningitis and controls, and between survivors and non-survivors, in order to provide insight into the host inflammatory response leading to poorer outcomes. DESIGN AND SETTING: Prospective case-control observational study in a tertiary hospital in Malawi. PARTICIPANTS: Children aged 2 months to 16 years with pneumococcal meningitis or pneumonia. METHODS: We used the human genome HGU133A Affymetrix array to explore differences in gene expression between cases with pneumococcal meningitis (n=12) and controls, and between HIV-infected and uninfected cases, and validated gene expression profiles for 34 genes using real-time quantitative PCR (RT-qPCR) in an independent set of cases with IPD (n=229) and controls (n=13). Pathway analysis was used to explore genes differentially expressed. RESULTS: Irrespective of underlying HIV infection, cases showed significant upregulation compared with controls of the following: S100 calcium-binding protein A12 (S100A12); vanin-1 (VNN1); arginase, liver (ARG1); matrix metallopeptidase 9 (MMP9); annexin A3 (ANXA3); interleukin 1 receptor, type II (IL1R2); CD177 molecule (CD177); endocytic adaptor protein (NUMB) and S100 calcium-binding protein A9 (S100A9), cytoskeleton-associated protein 4 (CKAP4); and glycogenin 1 (GYG1). RT-qPCR confirmed differential expression in keeping with microarray results. There was no differential gene expression in HIV-infected compared with HIV-uninfected cases, but there was significant upregulation of folate receptor 3 (FOLR3), S100A12 in survivors compared with non-survivors. CONCLUSION: Children with IPD demonstrated increased expression in genes regulating immune activation, oxidative stress, leucocyte adhesion and migration, arginine metabolism, and glucocorticoid receptor signalling.

SpeedyGenes: Exploiting an Improved Gene Synthesis Method for the Efficient Production of Synthetic Protein Libraries for Directed Evolution.

Gene synthesis is a fundamental technology underpinning much research in the life sciences. In particular, synthetic biology and biotechnology utilize gene synthesis to assemble any desired DNA sequence, which can then be incorporated into novel parts and pathways. Here, we describe SpeedyGenes, a gene synthesis method that can assemble DNA sequences with greater fidelity (fewer errors) than existing methods, but that can also be used to encode extensive, statistically designed sequence variation at any position in the sequence to create diverse (but accurate) variant libraries. We summarize the integrated use of GeneGenie to design DNA and oligonucleotide sequences, followed by the procedure for assembling these accurately and efficiently using SpeedyGenes.

Fragment-Based Approach to the Development of an Orally Bioavailable Lactam Inhibitor of Lipoprotein-Associated Phospholipase A2 (Lp-PLA2).

Lp-PLA2 has been explored as a target for a number of inflammation associated diseases, including cardiovascular disease and dementia. This article describes the discovery of a new fragment derived chemotype that interacts with the active site of Lp-PLA2. The starting fragment hit was discovered through an X-ray fragment screen and showed no activity in the bioassay (IC50 > 1 mM). The fragment hit was optimized using a variety of structure-based drug design techniques, including virtual screening, fragment merging, and improvement of shape complementarity. A novel series of Lp-PLA2 inhibitors was generated with low lipophilicity and a promising pharmacokinetic profile.