Structural analysis of a holoenzyme complex of mouse dihydrofolate reductase with NADPH and a ternary complex with the potent and selective inhibitor 2,4-diamino-6-(2′-hydroxydibenz[b,f]azepin-5-yl)methylpteridine

The structures of mouse DHFR holo enzyme and a ternary complex with NADPH and a potent inhibitor are described

Targeting species specific amino acid residues: Design, synthesis and biological evaluation of 6-substituted pyrrolo[2,3-d]pyrimidines as dihydrofolate reductase inhibitors and potential anti-opportunistic infection agents

To combine the potency of trimetrexate (TMQ) or piritrexim (PTX) with the species selectivity of trimethoprim (TMP), target based design was carried out with the X-ray crystal structure of human dihydrofolate reductase (hDHFR) and the homology model of Pneumocystis jirovecii DHFR (pjDHFR). Using variation of amino acids such as Met33/Phe31 (in pjDHFR/hDHFR) that affect the binding of inhibitors due to their distinct positive or negative steric effect at the active binding site of the inhibitor, we designed a series of substituted-pyrrolo[2,3-d]pyrimidines. The best analogs displayed better potency (IC50) than PTX and high selectivity for pjDHFR versus hDHFR, with 4 exhibiting a selectivity for pjDHFR of 24-fold

Evidence for the adaptation of protein pH-dependence to subcellular pH

<p>Abstract</p> <p>Background</p> <p>The availability of genome sequences, and inferred protein coding genes, has led to several proteome-wide studies of isoelectric points. Generally, isoelectric points are distributed following variations on a biomodal theme that originates from the predominant acid and base amino acid sidechain pKas. The relative populations of the peaks in such distributions may correlate with environment, either for a whole organism or for subcellular compartments. There is also a tendency for isoelectric points averaged over a subcellular location to not coincide with the local pH, which could be related to solubility. We now calculate the correlation of other pH-dependent properties, calculated from 3D structure, with subcellular pH.</p> <p>Results</p> <p>For proteins with known structure and subcellular annotation, the predicted pH at which a protein is most stable, averaged over a location, gives a significantly better correlation with subcellular pH than does isoelectric point. This observation relates to the cumulative properties of proteins, since maximal stability for individual proteins follows the bimodal isoelectric point distribution. Histidine residue location underlies the correlation, a conclusion that is tested against a background of proteins randomised with respect to this feature, and for which the observed correlation drops substantially.</p> <p>Conclusion</p> <p>There exists a constraint on protein pH-dependence, in relation to the local pH, that is manifested in the pKa distribution of histidine sub-proteomes. This is discussed in terms of protein stability, pH homeostasis, and fluctuations in proton concentration.</p

Next-Generation Sequencing Reveals Recent Horizontal Transfer of a DNA Transposon between Divergent Mosquitoes

Horizontal transfer of genetic material between complex organisms often involves transposable elements (TEs). For example, a DNA transposon mariner has been shown to undergo horizontal transfer between different orders of insects and between different phyla of animals. Here we report the discovery and characterization of an ITmD37D transposon, MJ1, in Anopheles sinensis. We show that some MJ1 elements in Aedes aegypti and An. sinensis contain intact open reading frames and share nearly 99% nucleotide identity over the entire transposon, which is unexpectedly high given that these two genera had diverged 145–200 million years ago. Chromosomal hybridization and TE-display showed that MJ1 copy number is low in An. sinensis. Among 24 mosquito species surveyed, MJ1 is only found in Ae. aegypti and the hyrcanus group of anopheline mosquitoes to which An. sinensis belongs. Phylogenetic analysis is consistent with horizontal transfer and provides the basis for inference of its timing and direction. Although report of horizontal transfer of DNA transposons between higher eukaryotes is accumulating, our analysis is one of a small number of cases in which horizontal transfer of nearly identical TEs among highly divergent species has been thoroughly investigated and strongly supported. Horizontal transfer involving mosquitoes is of particular interest because there are ongoing investigations of the possibility of spreading pathogen-resistant genes into mosquito populations to control malaria and other infectious diseases. The initial indication of horizontal transfer of MJ1 came from comparisons between a 0.4x coverage An. sinensis 454 sequence database and available TEs in mosquito genomes. Therefore we have shown that it is feasible to use low coverage sequencing to systematically uncover horizontal transfer events. Expanding such efforts across a wide range of species will generate novel insights into the relative frequency of horizontal transfer of different TEs and provide the evolutionary context of these lateral transfer events

pMHC affinity controls duration of CD8+ T cell&-DCinteractions and imprints timing of effector differentiationversus expansion

During adaptive immune responses, CD8+ T cells with low TCR affinities are released early into the circulation before high-affinityclones become dominant at later time points. How functional avidity maturation is orchestrated in lymphoid tissue andhow low-affinity cells contribute to host protection remains unclear. In this study, we used intravital imaging of reactive lymphnodes (LNs) to show that T cells rapidly attached to dendritic cells irrespective of TCR affinity, whereas one day later, theduration of these stable interactions ceased progressively with lowering peptide major histocompatibility complex (pMHC)affinity. This correlated inversely BATF (basic leucine zipper transcription factor, ATF-like) and IRF4 (interferon-regulatedfactor 4) induction and timing of effector differentiation, as low affinity&-primed T cells acquired cytotoxic activity earlierthan high affinity&-primed ones. After activation, low-affinity effector CD8+ T cells accumulated at efferent lymphatic vesselsfor egress, whereas high affinity&-stimulated CD8+ T cells moved to interfollicular regions in a CXCR3-dependent manner forsustained pMHC stimulation and prolonged expansion. The early release of low-affinity effector T cells led to rapid target cellelimination outside reactive LNs. Our data provide a model for affinity-dependent spatiotemporal orchestration of CD8+ T cellactivation inside LNs leading to functional avidity maturation and uncover a role for low-affinity effector T cells during earlymicrobial containment.This work was supported by a Novartis Research grant (to A.J. Ozga) and Swiss National Science Foundation grants (31003A_135649 and CR23I3_156234 to J.V. Stein and CRS II3_141918 to J.V. Stein and J. Sharpe). J. Ripoll acknowledges support from European Commission FP7 Career Integration Grants (HIGH-THROUGHPUT TOMO), European Commission FP7 Marie Curie Actions (grant 2PM), and the Ministerio de Economía y Competitividad (grant FIS2013-41802-R). J. Sharpe acknowledges support from the Ministerio de Economía y Competitividad, Centro de Excelencia Severo Ochoa 2013–2017 (grant SEV-2012-0208)

Large expert-curated database for benchmarking document similarity detection in biomedical literature search

Document recommendation systems for locating relevant literature have mostly relied on methods developed a decade ago. This is largely due to the lack of a large offline gold-standard benchmark of relevant documents that cover a variety of research fields such that newly developed literature search techniques can be compared, improved and translated into practice. To overcome this bottleneck, we have established the RElevant LIterature SearcH consortium consisting of more than 1500 scientists from 84 countries, who have collectively annotated the relevance of over 180 000 PubMed-listed articles with regard to their respective seed (input) article/s. The majority of annotations were contributed by highly experienced, original authors of the seed articles. The collected data cover 76% of all unique PubMed Medical Subject Headings descriptors. No systematic biases were observed across different experience levels, research fields or time spent on annotations. More importantly, annotations of the same document pairs contributed by different scientists were highly concordant. We further show that the three representative baseline methods used to generate recommended articles for evaluation (Okapi Best Matching 25, Term Frequency-Inverse Document Frequency and PubMed Related Articles) had similar overall performances. Additionally, we found that these methods each tend to produce distinct collections of recommended articles, suggesting that a hybrid method may be required to completely capture all relevant articles. The established database server located at https://relishdb.ict.griffith.edu.au is freely available for the downloading of annotation data and the blind testing of new methods. We expect that this benchmark will be useful for stimulating the development of new powerful techniques for title and title/abstract-based search engines for relevant articles in biomedical research.Peer reviewe

Finishing the euchromatic sequence of the human genome

The sequence of the human genome encodes the genetic instructions for human physiology, as well as rich information about human evolution. In 2001, the International Human Genome Sequencing Consortium reported a draft sequence of the euchromatic portion of the human genome. Since then, the international collaboration has worked to convert this draft into a genome sequence with high accuracy and nearly complete coverage. Here, we report the result of this finishing process. The current genome sequence (Build 35) contains 2.85 billion nucleotides interrupted by only 341 gaps. It covers ∼99% of the euchromatic genome and is accurate to an error rate of ∼1 event per 100,000 bases. Many of the remaining euchromatic gaps are associated with segmental duplications and will require focused work with new methods. The near-complete sequence, the first for a vertebrate, greatly improves the precision of biological analyses of the human genome including studies of gene number, birth and death. Notably, the human enome seems to encode only 20,000-25,000 protein-coding genes. The genome sequence reported here should serve as a firm foundation for biomedical research in the decades ahead

Repent Get Saved

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Structural analysis of Pneumocystis carinii and human DHFR complexes with NADPH and a series of five potent 6-[5′-(ω-carboxyalkoxy)benzyl]pyrido[2,3-d]pyrimidine derivatives

These data reveal that the ethyl esters of a series of 5′-(ω-carboxyalkoxy)benzylpyrido[2,3-d]pyrimidines have flexible side-chain conformations that do not optimize interactions with Arg75 in pcDHFR. Also, a novel conformation not reported in other DHFR structures was observed for one conformer of one of the compounds that had a disordered side chain