When a glimpse is enough: Partial mimicry of jumping spiders by insects.

Many flies and moths mimic the frontal appearance of jumping spiders. This type of mimicry, which we term as partial mimicry, can be distinguished from Batesian mimicry since the mimic has spider resembling patterns only in certain parts of the body, and not the entire body. The presence of spider-like patterns is obvious only at certain angles suggesting that the mimic is frequently targeted by its predators from particular angles. We tested this hypothesis using Deep Convolutional Neural Networks (DCNNs). First, we trained the network on images of forward facing jumping spiders, where features such as the large principal eyes, small lateral eyes and outstretched legs were evident. Then we tested the classifier on images of jumping spider mimicking flies and moths. A probability value according to the likelihood of the image being a jumping spider or not was assigned by the classifier. We show that the classifier was more likely to misidentify mimicking flies and moths as jumping spiders, but that this probability varied according to the species tested. We further tested it on images of flies from different angles and by taking into consideration the visual acuity of potential predators. Our results suggest that neural networks can be efficient tools for testing evolutionary hypotheses, and that partial mimicry may be a result of the effect of the signaling angle and orientation of the mimics in combination with the likelihood that predators may depend on cognitive shortcuts to identify insects as prey. Further experiments incorporating the properties of the visual system of predators (such as vision in ultraviolet) would result in a better understanding of the evolution of partial mimicry.

Collaborative Study to Validate Purity Determination by 1H Quantitative NMR Spectroscopy by Using Internal Calibration Methodology.

NMR spectroscopy has recently been utilized to determine the absolute amounts of organic molecules with metrological traceability since signal intensity is directly proportional to the number of each nucleus in a molecule. The NMR methodology that uses hydrogen nucleus (1H) to quantify chemicals is called quantitative 1H-NMR (1H qNMR). The quantitative method using 1H qNMR for determining the purity or content of chemicals has been adopted into some compendial guidelines and official standards. However, there are still few reports in the literature regarding validation of 1H qNMR methodology. Here, we coordinated an international collaborative study to validate a 1H qNMR based on the use of an internal calibration methodology. Thirteen laboratories participated in this study, and the purities of three samples were individually measured using 1H qNMR method. The three samples were all certified via conventional primary methods of measurement, such as butyl p-hydroxybenzoate Japanese Pharmacopeia (JP) reference standard certified by mass balance; benzoic acid certified reference material (CRM) certified by coulometric titration; fludioxonil CRM certified by a combination of freezing point depression method and 1H qNMR. For each sample, 1H qNMR experiments were optimized before quantitative analysis. The results showed that the measured values of each sample were equivalent to the corresponding reference labeled value. Furthermore, assessment of these 1H qNMR data using the normalized error, En-value, concluded that statistically 1H qNMR has the competence to obtain the same quantification performance and accuracy as the conventional primary methods of measurement.

Enabling adoption of 2D-NMR for the higher order structure assessment of monoclonal antibody therapeutics.

The increased interest in using monoclonal antibodies (mAbs) as a platform for biopharmaceuticals has led to the need for new analytical techniques that can precisely assess physicochemical properties of these large and very complex drugs for the purpose of correctly identifying quality attributes (QA). One QA, higher order structure (HOS), is unique to biopharmaceuticals and essential for establishing consistency in biopharmaceutical manufacturing, detecting process-related variations from manufacturing changes and establishing comparability between biologic products. To address this measurement challenge, two-dimensional nuclear magnetic resonance spectroscopy (2D-NMR) methods were introduced that allow for the precise atomic-level comparison of the HOS between two proteins, including mAbs. Here, an inter-laboratory comparison involving 26 industrial, government and academic laboratories worldwide was performed as a benchmark using the NISTmAb, from the National Institute of Standards and Technology (NIST), to facilitate the translation of the 2D-NMR method into routine use for biopharmaceutical product development. Two-dimensional 1H,15N and 1H,13C NMR spectra were acquired with harmonized experimental protocols on the unlabeled Fab domain and a uniformly enriched-15N, 20%-13C-enriched system suitability sample derived from the NISTmAb. Chemometric analyses from over 400 spectral maps acquired on 39 different NMR spectrometers ranging from 500 MHz to 900 MHz demonstrate spectral fingerprints that are fit-for-purpose for the assessment of HOS. The 2D-NMR method is shown to provide the measurement reliability needed to move the technique from an emerging technology to a harmonized, routine measurement that can be generally applied with great confidence to high precision assessments of the HOS of mAb-based biotherapeutics.

NMR characterization of an oligonucleotide model of the miR-21 pre-element.

We have used NMR spectroscopy to characterize an oligonucleotide stem loop structure based on the pre-element of an oncogenic microRNA, miR-21. This predicted stem-loop structure is cleaved from the precursor of miR-21 (pre-miR-21) by the nuclease Dicer. It is also a critical feature recognized by the protein complex that converts the primary transcript (pri-miR-21) into the pre-miRNA. The secondary structure of the native sequence is poorly defined by NMR due to rapid exchange of imino protons with solvent; however, replacement of two adjacent putative G•U base pairs with G•C base pairs retains the conformation of the hairpin observed by chemical probing and stabilizes it sufficiently to observe most of the imino proton resonances of the molecule. The observed resonances are consistent with the predicted secondary structure. In addition, a peak due to a loop uridine suggests an interaction between it and a bulged uridine in the stem. Assignment of non-exchangeable proton resonances and characterization of NOEs and coupling constants allows inference of the following features of the structure: extrahelicity of a bulged adenosine, deviation from A-form geometry in a base-paired stem, and consecutive stacking of the adenosines in the 5' side of the loop, the guanosine of the closing base pair, and a cross-strand adenosine. Modeling of the structure by restrained molecular dynamics suggests a basis for the interaction between the loop uridine, the bulged uridine in the stem, and an A•U base pair in the stem.

Assessment of higher order structure comparability in therapeutic proteins using nuclear magnetic resonance spectroscopy.

In this work, we applied nuclear magnetic resonance (NMR) spectroscopy to rapidly assess higher order structure (HOS) comparability in protein samples. Using a variation of the NMR fingerprinting approach described by Panjwani et al. [2010. J Pharm Sci 99(8):3334-3342], three nonglycosylated proteins spanning a molecular weight range of 6.5-67 kDa were analyzed. A simple statistical method termed easy comparability of HOS by NMR (ECHOS-NMR) was developed. In this method, HOS similarity between two samples is measured via the correlation coefficient derived from linear regression analysis of binned NMR spectra. Applications of this method include HOS comparability assessment during new product development, manufacturing process changes, supplier changes, next-generation products, and the development of biosimilars to name just a few. We foresee ECHOS-NMR becoming a routine technique applied to comparability exercises used to complement data from other analytical techniques.

Improvement of resolution for brain coupled metabolites by optimized (1)H MRS at 7T.

Resolution enhancement for glutamate (Glu), glutamine (Gln) and glutathione (GSH) in the human brain by TE-optimized point-resolved spectroscopy (PRESS) at 7 T is reported. Sub-TE dependences of the multiplets of Glu, Gln, GSH, γ-aminobutyric acid (GABA) and N-acetylaspartate (NAA) at 2.2-2.6 ppm were investigated with density matrix simulations, incorporating three-dimensional volume localization. The numerical simulations indicated that the C4-proton multiplets can be completely separated with (TE(1), TE(2)) = (37, 63) ms, as a result of a narrowing of the multiplets and suppression of the NAA 2.5 ppm signal. Phantom experiments reproduced the signal yield and lineshape from simulations within experimental errors. In vivo tests of optimized PRESS were conducted on the prefrontal cortex of six healthy volunteers. In spectral fitting by LCModel, Cramér-Rao lower bounds (CRLBs) of Glu, Gln and GSH were 2 ± 1, 5 ± 1 and 6 ± 2 (mean ± SD), respectively. To evaluate the performance of the optimized PRESS method under identical experimental conditions, stimulated-echo spectra were acquired with (TE, TM) = (14, 37) and (74, 68) ms. The CRLB of Glu was similar between PRESS and short-TE stimulated-echo acquisition mode (STEAM), but the CRLBs of Gln and GSH were lower in PRESS than in both STEAM acquisitions.

Molecular basis of coiled coil coactivator recruitment by the aryl hydrocarbon receptor nuclear translocator (ARNT).

The aryl hydrocarbon receptor nuclear translocator (ARNT) serves as the obligate heterodimeric partner for bHLH-PAS proteins involved in sensing and coordinating transcriptional responses to xenobiotics, hypoxia, and developmental pathways. Although its C-terminal transactivation domain is dispensable for transcriptional activation in vivo, ARNT has recently been shown to use its N-terminal bHLH and/or PAS domains to interact with several transcriptional coactivators that are required for transcriptional initiation after xenobiotic or hypoxic cues. Here we show that ARNT uses a single PAS domain to interact with two coiled coil coactivators, TRIP230 and CoCoA. Both coactivators interact with the same interface on the ARNT PAS-B domain, located on the opposite side of the domain used to associate with the analogous PAS domain on its heterodimeric bHLH-PAS partner HIF-2alpha. Using NMR and biochemical studies, we identified the ARNT-interacting motif of one coactivator, TRIP230 as an LXXLL-like nuclear receptor box. Mutation of this motif and proximal sequences disrupts the interaction with ARNT PAS-B. Identification of this ARNT-coactivator interface illustrates how ARNT PAS-B is used to form critical interactions with both bHLH-PAS partners and coactivators that are required for transcriptional responses.

Signal transduction pathway of TonB-dependent transporters.

Transcription of the ferric citrate import system is regulated by ferric citrate binding to the outer membrane transporter FecA. A signal indicating transporter occupancy is relayed across the outer membrane to energy-transducing and regulatory proteins embedded in the cytoplasmic membrane. Because transcriptional activation is not coupled to ferric citrate import, an allosteric mechanism underlies this complex signaling mechanism. Using evolution-based statistical analysis we have identified a sparse but structurally connected network of residues that links distant functional sites in FecA. Functional analyses of these positions confirm their involvement in the mechanism that regulates transcriptional activation in response to ferric citrate binding at the cell surface. This mechanism appears to be conserved and provides the structural basis for the allosteric signaling of TonB-dependent transporters.

NMR structures of the selenoproteins Sep15 and SelM reveal redox activity of a new thioredoxin-like family.

Selenium has significant health benefits, including potent cancer prevention activity and roles in immune function and the male reproductive system. Selenium-containing proteins, which incorporate this essential micronutrient as selenocysteine, are proposed to mediate the positive effects of dietary selenium. Presented here are the solution NMR structures of the selenoprotein SelM and an ortholog of the selenoprotein Sep15. These data reveal that Sep15 and SelM are structural homologs that establish a new thioredoxin-like protein family. The location of the active-site redox motifs within the fold together with the observed localized conformational changes after thiol-disulfide exchange and measured redox potential indicate that they have redox activity. In mammals, Sep15 expression is regulated by dietary selenium, and either decreased or increased expression of this selenoprotein alters redox homeostasis. A physiological role for Sep15 and SelM as thiol-disulfide oxidoreductases and their contribution to the quality control pathways of the endoplasmic reticulum are discussed.

Identification of small-molecule antagonists that inhibit an activator: coactivator interaction.

Phosphorylation of the cAMP response element binding protein (CREB) at Ser-133 in response to hormonal stimuli triggers cellular gene expression via the recruitment of the histone acetylase coactivator paralogs CREB binding protein (CBP) and p300 to the promoter. The NMR structure of the CREB:CBP complex, using relevant interaction domains called KID and KIX, respectively, reveals a shallow hydrophobic groove on the surface of KIX that accommodates an amphipathic helix in phospho (Ser-133) KID. Using an NMR-based screening approach on a preselected small-molecule library, we identified several compounds that bind to different surfaces on KIX. One of these, KG-501 (2-naphthol-AS-E-phosphate), targeted a surface distal to the CREB binding groove that includes Arg-600, a residue that is required for the CREB:CBP interaction. When added to live cells, KG-501 disrupted the CREB: CBP complex and attenuated target gene induction in response to cAMP agonist. These results demonstrate the ability of small molecules to interfere with second-messenger signaling cascades by inhibiting specific protein-protein interactions in the nucleus.

Acciones para restablecer la permeabilidad de la venoclisis por personal de enfermería / Actions performed by the nursing personal for to restore the permeability of the venoclisis

Introducción: La enfermería requiere dejar atrás todas aquellas acciones no establecidas por los organismos rectores de la misma y que cotidianamente realiza sin reflexionar y sin ningún sustento teórico-científico, incrementando con ello la posibilidad de complicaciones para el paciente. Objetivo: Identificar las acciones enfermería del Hospital Regional Universitario para restablecer la permeabilidad de venoclisis. Metodología: Estudio descriptivo, transversal con muestreo no probabilística por conveniencia con 34 enfermeras (os). Se efectuó estudio de sombra, cegado en donde se comparara las acciones referidas con las realizadas durante las acciones para permeabilizar las venoclisis. Análisis mediante estadística descriptiva. Resultados: El 11.8% refirió utilizar un bolo de infusión de solución fisiológica y se observó que lo realiza 41%; 11.8% refirió torcer el tubo del equipo y 20.6% lo realizó; 11.8% refirió observar sielequipoestabadob!adoysolo3% lo efectuó; 11.8% menciono verificar altura adecuada del frasco de solución y lo realizó 3%; 7% afirmó aplicar heparina y nadie lo realizó. Conclusiones: La práctica de enfermería debe basarse en la observancia de criterios éticos que protejan la integridad del paciente, ante una venoclisis obstruida es conveniente que se realicen primeramente medidas de observación antes que las de acción o manipulación.

Actions performed by the nursing personal for to restore the permeability of the venoclisis Introduction: The nursing needs to leave behind all those actions not established by the governing organisms of the same and that everyday perform without thinking and any theoretical-scientific support, increasing with it the possibility of complications to the patient. Objetive: To identify the actions performed by the nursing personal of the Regional University Hospital to restore the permeability of venoclisis. Methodology: Descriptive transverse study with sampling not probabilistic for convenience with 34 nurses. There was effected an study of shade blinded where the refered actions were compared with the performed during the actions to permeate the venoclisis. Analysis by descriptive statistics was used. Results: 11.8% refered to use a bolus of physiological infusion and was observed that 41 % realized it; 11.8% refered to twist the equipment tube and 20.6% realized it; 11.8% refered to observe if the equipment was doubled and only 3% carried out it; 11.8% refered to check the appropriate height of the saline solution and 3% realized it; 7% affirmed to apply heparin and nobody carried out it. Conclusions: In presence of blocked venoclisis, it is appropriate to perform procedures of observation before those of action or manipulation. The practice of nursing must be based on the observance of ethical criteria that protect the integrity of the patient.

Structure and interactions of PAS kinase N-terminal PAS domain: model for intramolecular kinase regulation.

PAS domains are sensory modules in signal-transducing proteins that control responses to various environmental stimuli. To examine how those domains can regulate a eukaryotic kinase, we have studied the structure and binding interactions of the N-terminal PAS domain of human PAS kinase using solution NMR methods. While this domain adopts a characteristic PAS fold, two regions are unusually flexible in solution. One of these serves as a portal that allows small organic compounds to enter into the core of the domain, while the other binds and inhibits the kinase domain within the same protein. Structural and functional analyses of point mutants demonstrate that the compound and ligand binding regions are linked, suggesting that the PAS domain serves as a ligand-regulated switch for this eukaryotic signaling system.