User-friendly Protocol Research Articles

LARMD: integration of bioinformatic resources to profile ligand-driven protein dynamics with a case on the activation of estrogen receptor.

Protein dynamics is central to all biological processes, including signal transduction, cellular regulation and biological catalysis. Among them, in-depth exploration of ligand-driven protein dynamics contributes to an optimal understanding of protein function, which is particularly relevant to drug discovery. Hence, a wide range of computational tools have been designed to investigate the important dynamic information in proteins. However, performing and analyzing protein dynamics is still challenging due to the complicated operation steps, giving rise to great difficulty, especially for nonexperts. Moreover, there is a lack of web protocol to provide online facility to investigate and visualize ligand-driven protein dynamics. To this end, in this study, we integrated several bioinformatic tools to develop a protocol, named Ligand and Receptor Molecular Dynamics (LARMD, http://chemyang.ccnu.edu.cn/ccb/server/LARMD/ and http://agroda.gzu.edu.cn:9999/ccb/server/LARMD/), for profiling ligand-driven protein dynamics. To be specific, estrogen receptor (ER) was used as a case to reveal ERβ-selective mechanism, which plays a vital role in the treatment of inflammatory diseases and many types of cancers in clinical practice. Two different residues (Ile373/Met421 and Met336/Leu384) in the pocket of ERβ/ERα were the significant determinants for selectivity, especially Met336 of ERβ. The helix H8, helix H11 and H7-H8 loop influenced the migration of selective agonist (WAY-244). These computational results were consistent with the experimental results. Therefore, LARMD provides a user-friendly online protocol to study the dynamic property of protein and to design new ligand or site-directed mutagenesis.

Capacity and willingness of farmers and citizen scientists to monitor crop pollinators and pollination services

Insects pollinate many globally important crops and therefore rapid and effective means to measure crop pollinators and pollination are required to support national monitoring schemes and allow localised measurements of pollinator supply and demand to crops. We tested user-friendly protocols for assessing pollinators and pollination in crops to better understand the capacity and willingness of a group of farmers and citizen scientists to implement such techniques in the field. We asked the different recorder groups including farmers and agronomists, non-expert volunteers and experienced researchers to complete three pollinator and pollination service assessment techniques: transect walks, pan trapping and pollinator exclusion and supplementary pollination. Recorders provided feedback on each method through a questionnaire and the data collected using different methods were compared. Our volunteer members of the public, and farmers and agronomists were able to implement all assessment techniques in apple, bean and oilseed rape fields. The experienced researchers and volunteer members of the public were more willing to record bumblebees to species level on transects than the farmers and agronomists. There was also a significant interaction between recorder and crop type for certain insect taxa demonstrating that in certain crops some taxa may be easier to record than others. All our recorder groups found transects and pan traps straightforward and enjoyable to implement. Our non-expert volunteers were willing to use pollinator exclusion and supplementary pollination techniques as part of a wider scheme, the farmers and agronomists who implemented the technique were less positive about applying this method more widely. We have demonstrated that volunteer recorders, including farmers and agronomists, can be engaged and are able to implement methods to assess pollinators and pollination, although additional training is necessary to ensure accurate species data collection. For the more direct and time consuming measures of pollination service, both training and additional support may be needed, particularly for farmers. The tools developed and tested here will be valuable for wider pollinator monitoring schemes and for integration into standard agronomic practices.

QresFEP: An Automated Protocol for Free Energy Calculations of Protein Mutations in Q.

Predicting the effect of single-point mutations on protein stability or protein-ligand binding is a major challenge in computational biology. Free energy calculations constitute the most rigorous approach to this problem, though the estimation of converged values for amino acid mutations remains challenging. To overcome this limitation, we developed tailored protocols to calculate free energy shifts associated with single-point mutations. We herein describe the QresFEP protocol, which includes an extension of our recent protocols to cover all amino acids mutations, based on the latest versions of the OPLS-AA force field. QresFEP is implemented in an application programming interface framework and the graphic interface QGui, for the molecular dynamics software Q. The complete protocol is benchmarked in several model systems, optimizing a number of sampling parameters and the implementation of Zwanzig's exponential formula and Bennet's acceptance ratio methods. QresFEP shows an excellent performance on estimating the hydration free energies of amino acid side-chain mimics, including their charged analogues. We also examined its performance on a protein-ligand binding problem of pharmaceutical relevance, the antagonism of neuropeptide Y1 G protein-coupled receptor. Here, the calculations show very good agreement with the experimental effect of 16 mutations on the binding of antagonists BIBP3226, in line with our recent applications in this field. Finally, the characterization of 43 mutations of T4-lysozyme reveals the capacity of our protocol to assess variations of the thermal stability of proteins, achieving a similar performance to alternative free energy perturbation (FEP) approaches. In summary, QresFEP is a robust, versatile, and user-friendly computational FEP protocol to examine biochemical effects of single-point mutations with high accuracy.

Definition and validation of a custom protocol to detect miRNAs in the spent media after blastocyst culture: searching for biomarkers of implantation

Can miRNAs be reliably detected in the spent blastocyst media (SBM) after IVF as putative biomarkers of the implantation potential of euploid embryos? Adjustment of the data for blastocyst quality and the day of full-expansion hinders the predictive power of a fast, inexpensive, reproducible and user-friendly protocol based on the detection of 10 selected miRNAs from SBM. Euploidy represents so far the strongest predictor of blastocyst competence. Nevertheless, ~50% of the euploid blastocysts fail to implant. Several studies across the years have suggested that a dialogue exists between the embryo and the endometrium aimed at the establishment of a pregnancy. MicroRNAs have been proposed as mediators of such a dialogue and investigated in this respect. Several expensive, time-consuming and complex protocols have been adopted and promising results have been produced, but conclusive evidence from large clinical studies is missing. This study was conducted in two phases from September 2015 to December 2017. In Phase 1, the human blastocyst miRNome profile was defined from the inner cell mass (ICM) and the corresponding whole-trophectoderm (TE) of six donated blastocysts. Two different protocols were adopted to this end. In parallel, 6 pools of 10 SBM each were run (3 from only implanted euploid blastocysts, IEBs; and 3 from only not-implanted euploid blastocysts, not-IEBs). A fast, inexpensive and user-friendly custom protocol for miRNA SBM profiling was designed. In Phase 2, 239 SBM from IEB and not-IEB were collected at three IVF centres. After 18 SBM from poor-quality blastocysts were excluded from the analysis, data from 107 SBM from not-IEB and 114 from IEB were produced through the previously developed custom protocol and compared. The data were corrected through logistic regressions. Donated blastocysts underwent ICM and whole-TE isolation. SBM were collected during IVF cycles characterized by ICSI, blastocyst culture in a continuous media, TE biopsy without zona pellucida opening in Day3, quantitative PCR (qPCR)-based aneuploidy testing and vitrified-warmed single euploid embryo transfer. Not-IEB and IEB were clustered following a negative pregnancy test and a live birth, respectively. The Taqman Low Density Array (TLDA) cards and the Exiqon microRNA human panel I+II qPCR analysis protocols were adopted to analyse the ICM and whole-TE. The latter was used also for SBM pools. A custom protocol and plate was then designed based on the Exiqon workflow, validated and finally adopted for SBM analysis in study Phase 2. This custom protocol allows the analysis of 10 miRNAs from 10 SBM in 3hours from sample collection to data inspection. The TLDA cards protocol involved a higher rate of false positive results (5.6% versus 2.8% with Exiqon). There were 44 miRNAs detected in the ICM and TE from both the protocols. One and 24 miRNAs were instead detected solely in the ICM and the TE, respectively. Overall, 29 miRNAs were detected in the pooled SBM: 8 only from not-IEB, 8 only from IEB and 13 from both. Most of them (N = 24/29, 82.7%) were also detected previously in both the ICM and TE with the Exiqon protocol; two miRNAs (N = 2/29, 6.9%) were previously detected only in the TE, and three (N = 3/29, 10.3%) were never detected previously. In study Phase 2, significant differences were shown between not-IEB and IEB in terms of both miRNA detection and relative quantitation. However, when the data were corrected for embryo morphology and day of full development (i.e. SBM collection), no significant association was confirmed. This study did not evaluate specifically exosomal miRNAs, thereby reducing the chance of identifying the functional miRNAs. Ex-vivo experiments are required to confirm the role of miRNAs in mediating the dialogue with endometrial cells, and higher throughput technologies need to be further evaluated for miRNA profiling from clinical SBM samples. Although no clinical predictive power was reported in this study, the absence of invasiveness related with SBM analysis and the evidence that embryonic genetic material can be reliably detected and analysed from SBM make this waste product of IVF an important source for further investigations aimed at improving embryo selection. This project has been financially supported by Merck KgaA (Darmstadt, Germany) with a Grant for Fertility Innovation (GFI) 2015. The authors have no conflict of interest to declare related with this study. None.

Toward Single-Organelle Lipidomics in Live Cells.

Detailed studies of lipids in biological systems, including their role in cellular structure, metabolism, and disease development, comprise an increasingly prominent discipline called lipidomics. However, the conventional lipidomics tools, such as mass spectrometry, cannot investigate lipidomes until they are extracted, and thus they cannot be used for probing the lipid distribution nor for studying in live cells. Furthermore, conventional techniques rely on the lipid extraction from relatively large samples, which averages the data across the cellular populations and masks essential cell-to-cell variations. Further advancement of the discipline of lipidomics critically depends on the capability of high-resolution lipid profiling in live cells and, potentially, in single organelles. Here we report a micro-Raman assay designed for single-organelle lipidomics. We demonstrate how Raman microscopy can be used to measure the local intracellular biochemical composition and lipidome hallmarks-lipid concentration and unsaturation level, cis/trans isomer ratio, sphingolipids and cholesterol levels in live cells-with a sub-micrometer resolution, which is sufficient for profiling of subcellular structures. These lipidome data were generated by a newly developed biomolecular component analysis software, which provides a shared platform for data analysis among different research groups. We outline a robust, reliable, and user-friendly protocol for quantitative analysis of lipid profiles in subcellular structures. This method expands the capabilities of Raman-based lipidomics toward the analysis of single organelles within either live or fixed cells, thus allowing an unprecedented measure of organellar lipid heterogeneity and opening new quantitative ways to study the phenotypic variability in normal and diseased cells.

Compliance and Practical Utility of Continuous Wearing of activPAL™ in Adolescents.

The aim of this study was to examine the factors that influence compliance and practical utility of a continuous wear protocols for activPAL™ among adolescents. Seven hundred and fifty-five (11-18y; 50.6% girls) students wore the waterproof activPAL™ for 7 consecutive days. The effects of factors such as weather and practical strategies on compliance were assessed. Students were asked to note reasons for removing it in a log. After the 7-day period, students anonymously completed a practical utility questionnaire. The final sample used to analyze compliance contained 588 available data points; 72.4% met the validity criteria, which were ≥4 valid days. Rainfall was inversely associated with total wear time, whereas using alcohol pads and cartoon stickers during the application were positively associated with total wear time. Sweating (25.2%) and skin irritation (39.0%) were the most reasons for 290 removal episodes by 235 students. The 131 questionnaires showed that 80.1% regarded the continuous wear period as too long and encountered problems, and 55% would rather not wear it again. Rainy weather affected girls' compliance with the continuous wear protocol for activPAL™. Skin irritation and sweat-induced inadvertent drops caused removal. Future studies should investigate more user-friendly protocols.

Single-site glycine-specific labeling of proteins

Labeling of native proteins invites interest from diverse segments of science. However, there remains the significant unmet challenge in precise labeling at a single site of a protein. Here, we report the site-specific labeling of natural or easy-to-engineer N-terminus Gly in proteins with remarkable efficiency and selectivity. The method generates a latent nucleophile from N-terminus imine that reacts with an aldehyde to deliver an aminoalcohol under physiological conditions. It differentiates N-Gly as a unique target amongst other proteinogenic amino acids. The method allows single-site labeling of proteins in isolated form and extends to lysed cells. It administers an orthogonal aldehyde group primed for late-stage tagging with an affinity tag, 19F NMR probe, and a fluorophore. A user-friendly protocol delivers analytically pure tagged proteins. The mild reaction conditions do not alter the structure and function of the protein. The cellular uptake of fluorophore-tagged insulin and its ability to activate the insulin-receptor mediated signaling remains unperturbed.

Rapid and selective detection of Fe (III) by using a smartphone-based device as a portable detector and hydroxyl functionalized metal-organic frameworks as the fluorescence probe

Here, a label-free fluorescent sensor was developed for detection Fe (III) by utilizing the quenching effect of Fe (III) on the fluorescence of the hydroxyl functionalized metal-organic framework MIL-53(Fe)-(OH)2, which was synthesized by using a one-step solvothermal method. The specific binding interaction between Fe (III) and hydroxyl facilitated the absorption of free Fe (III) to MIL-53(Fe)-(OH)2, which leads to rapid fluorescent intensity quenching effect. The potential quenching mechanism was proved to be the photo-induced electron transfer (PET) from electron-rich ligands of MIL-53(Fe)-(OH)2 to the half-filled 3d orbitals of free Fe (III) in the sample solution. For in-field applications, the fluorescence signal was detected rapidly by using a homemade 3D-printed, portable, and low-cost smartphone sensor. A commercial 365 nm UV LED light was adopted as the excitation light source, while the camera in a smartphone was utilized as the optical detector. The fluorescent signals obtained by using the smartphone sensor were in a good agreement with those obtained from a commercial microplate reader. Under the optimal assay conditions, the linear detection range of Fe (III) was 5.0–200 μM, and the limit of detection is 1.7 μM. This result is compatible with the commercial microplate reader. The developed method was successfully adopted to detect Fe (III) in human serum and environmental water samples with acceptable recovery values of 90–108.5%. The portable, low-cost, fast-response, user-friendly and sensitive fluorescent protocol based on a self-quenching fluorescent nanoprobe can be conducted at home or anywhere else without sophisticated instruments, showing a great application potential in clinical diagnosis, on-site environmental monitoring and healthcare at home.

Grain-size distribution unmixing using the R package EMMAgeo

Abstract. The analysis of grain-size distributions has a long tradition in Quaternary Science and disciplines studying Earth surface and subsurface deposits. The decomposition of multi-modal grain-size distributions into inherent subpopulations, commonly termed end-member modelling analysis (EMMA), is increasingly recognised as a tool to infer the underlying sediment sources, transport and (post-)depositional processes. Most of the existing deterministic EMMA approaches are only able to deliver one out of many possible solutions, thereby shortcutting uncertainty in model parameters. Here, we provide user-friendly computational protocols that support deterministic as well as robust (i.e. explicitly accounting for incomplete knowledge about input parameters in a probabilistic approach) EMMA, in the free and open software framework of R. In addition, and going beyond previous validation tests, we compare the performance of available grain-size EMMA algorithms using four real-world sediment types, covering a wide range of grain-size distribution shapes (alluvial fan, dune, loess and floodplain deposits). These were randomly mixed in the lab to produce a synthetic data set. Across all algorithms, the original data set was modelled with mean R2 values of 0.868 to 0.995 and mean absolute deviation (MAD) values of 0.06 % vol to 0.34 % vol. The original grain-size distribution shapes were modelled as end-member loadings with mean R2 values of 0.89 to 0.99 and MAD of 0.04 % vol to 0.17 % vol. End-member scores reproduced the original mixing ratios in the synthetic data set with mean R2 values of 0.68 to 0.93 and MAD of 0.1 % vol to 1.6 % vol. Depending on the validation criteria, all models provided reliable estimates of the input data, and each of the models exhibits individual strengths and weaknesses. Only robust EMMA allowed uncertainties of the end-members to be objectively estimated and expert knowledge to be included in the end-member definition. Yet, end-member interpretation should carefully consider the geological and sedimentological meaningfulness in terms of sediment sources, transport and deposition as well as post-depositional alteration of grain sizes. EMMA might also be powerful in other geoscientific contexts where the goal is to unmix sources and processes from compositional data sets.

Bacteria-Derived Recombinant Human ANGPTL8/Betatrophin Significantly Increases the Level of Triglyceride

ANGPTL8/Betatrophin has been implicated in the regulation of both glucose and triglyceride metabolism. However, its role in regulating glucose metabolism by promoting β cell proliferation remains controversial, and its physiological functions and molecular targets are largely unknown. Hence, it is of great importance to make recombinant protein and test its effects on β cell mass directly. In this study, the mature form gene of human ANGPTL8/betatrophin was obtained through chemical synthesis on to the vector pUCE, and the fusion protein was expressed in the Transetta (DE3)/pEASY-E2-betatrophin strain. The inclusion bodies were solubilized in urea and purified by Ni–NTA affinity chromatography. The yield of purified ANGPTL8/betatrophin was approximately 20 mg per liter of culture medium. In vitro studies revealed that the recombinant ANGPTL8/betatrophin had no proliferation effect on MIN6 cells but promoted TG levels in HepG2 cells. This method to generate bioactive ANGPTL8/betatrophin is a simple, practical and user-friendly protocol.

Ni-catalyzed Reductive Deaminative Arylation at sp3 Carbon Centers.

A Ni-catalyzed reductive deaminative arylation at unactivated sp3 carbon centers is described. This operationally simple and user-friendly protocol exhibits excellent chemoselectivity profile and broad substrate scope, thus complementing existing metal-catalyzed cross-coupling reactions to forge sp3 C-C linkages. These virtues have been assessed in the context of late-stage functionalization, hence providing a strategic advantage to reliably generate structure diversity with amine-containing drugs.

Hindering the illegal trade in dog and cat furs through a DNA-based protocol for species identification.

In Western countries dogs and cats are the most popular pets, and people are increasingly opposed to their rearing for the fur industry. In 2007, a Regulation of the European Union (EU) banned the use and trade of dog and cat furs, but an official analytical protocol to identify them as source species was not provided, and violations of law are still frequent in all Member States. In this paper we report on the development and validation of a simple and affordable DNA method for species detection in furs to use as an effective tool to combat illegal trade in fur products. A set of mitochondrial primers was designed for amplification of partial cytochrome b, control region and ND1 gene in highly degraded samples, like furs and pelts. Our amplification workflow involved the use of a non-specific primer pair to perform a first test to identify the species through sequencing, then the application of species-specific primer pairs to use in singleplex end-point PCRs as confirmation tests. The advantage of this two-step procedure is twofold: on the one hand it minimises the possibility of negative test results from degraded samples, since failure of amplification with a first set of primers can be offset by successful amplification of the second, and on the other it adds confidence and reliability to final authentication of species. All designed primers were validated on a reference collection of tissue samples, obtaining solid results in terms of specificity, sensitivity, repeatability and reproducibility. Application of the protocol on real caseworks from seized furs yielded successful results also from old and dyed furs, suggesting that age and chemical staining do not necessarily affect positive amplifications. Major pros of this approach are: (1) sensitive and informative primer sets for detection of species; (2) short PCR amplicons for the analysis of poor quality DNA; (3) binding primers that avoid contamination from human DNA; (4) user-friendly protocol for any laboratory equipped for analysis of low-copy-number DNA. Our molecular procedure proved to be a good starting point for enforcing the EU Regulation against dog and cat fur trade in forensic contexts where source attribution is essential to the assignment of responsibilities.

Production of soluble bioactive mouse leukemia inhibitory factor from Escherichia coli using MBP tag

Embryonic stem cells and induced pluripotent stem cells depend on one of cytokines called leukemia inhibitory factor (LIF) to retain their undifferentiated state and pluripotency. Nevertheless, further progresses of stem cell scientific investigation and its possible application are limited owing to the expense of commercial LIF. Here we introduced a simple, practical and high level expression of MBP-mouse LIF through Escherichia coli system which was bioactive. The mLIF cDNA was inserted into vector of pMAL-C2X in order to generate N-terminal MBP-mLIF recombinant proteins in the cytoplasm of Escherichia coli. MBP-mLIF as a soluble form was expressed. One-step purification through gravitational affinity chromatography was accomplished to acquire high purity (>92%) MBP-mLIF. The MBP-mLIF products specifically suppressed the growth of M1cells in a dose-dependent pattern. MBP-mLIF also was proved the ability to maintain the pluripotency of iPSCs. These outcomes revealed that the N-end MBP tags of the MBP-mLIF did not obstruct mLIF bioactivity. This method to generate recombinant MBP-mLIF is a simple, practical, economical and user-friendly protocol.

Cloud computing and big data: Technologies and applications

Cloud computing and big data: Technologies and applications

Simple and Practical Real-Time Analysis of Solid-Phase Reactions by Thin-Layer Chromatography

Solid-phase synthesis is a practical approach for simplifying the time-consuming and routine purification steps in the preparation of numerous naturally occurring molecules; however, studying such reactions is difficult due to the lack of a convenient monitoring method. By using thin-layer chromatography in conjunction with a photolabile linker on a resin, we developed a convenient and simple method for monitoring solid-phase reactions in real time by thin-layer chromatography. This method provides a user-friendly protocol for examining reaction conditions for solid-state syntheses.

Olefin-Stabilized Cobalt Nanoparticles for C=C, C=O, and C=N Hydrogenations.

The development of cobalt catalysts that combine easy accessibility and high selectivity constitutes a promising approach to the replacement of noble-metal catalysts in hydrogenation reactions. This report introduces a user-friendly protocol that avoids complex ligands, hazardous reductants, special reaction conditions, and the formation of highly unstable pre-catalysts. Reduction of CoBr2 with LiEt3 BH in the presence of alkenes led to the formation of hydrogenation catalysts that effected clean conversions of alkenes, carbonyls, imines, and heteroarenes at mild conditions (3 mol % cat., 2-10 bar H2 , 20-80 °C). Poisoning studies and nanoparticle characterization by TEM, EDX, and DLS supported the notion of a heterotopic catalysis mechanism.

Bisoma five element acupuncture

Bisoma five element acupuncture

Constructing a secure hacking-resistant IoT U-healthcare environment

There has been an increasing demand of work that required high level of computing power in IoT or establishment of server in IoT environment due to development of computing power. Excessive diagnosis has recently been a significant issue in the field of medicine. Excessive diagnosis issue is expected to be significantly reduced if establishing the easily configurable IoT server, communication with medical devices, and communication among hospitals by transmitting medical results to users. With the advancement of hacking technology, many hackers are interested in medical information and medical infrastructure, and the Information Security Management System (ISMS) is expanding in Korea. A number of medical ISMS projects are discussed, based on ISO 27791 standards derived from ISO 27001 standards. Large hospitals in South Korea have a variety of platforms that misrepresent personal information, and various platforms deal with sensitive medical information, but small and medium-sized hospitals use platforms that are not considered legal considerations.In this paper, we intend to establish a legal information sharing platform, which is legally considered legally limited and is responsible for the safety of the medical information.In the IoT server with limited resources, light-weighted and user-friendly protocols are frequently utilized over those that derive traffic and consume much resource. Under these circumstances, this study is intended to suggest IoT network that medical devices are able to create ubiquitous environment for users by utilizing simple service discovery protocol as a protocol provided without imposing much burden on limited resources and highly secured extensible messaging and presence protocol.

A General Copper/Manganese Cocatalyzed C-H Selenation of Arenes, Heteroarenes, and Alkenes under Air

Despite significant progress, copper-catalyzed, directed C−H activation to form C-heteroatom bond is a challenging problem due to strong chelation with the directing group and the incoming nucleophile. Therefore, stoichiometric copper salt was used which is not practical from industrial standpoint. Here we have developed a copper-manganese dual catalytic system for a general chelation-assisted C(sp2)-H selenation of benzamides and acrylamides. Interestingly, the acrylamides provided thermodynamically unfavourable (Z)-vinyl selenides with complete control of regio- and stereoselectivity. In addition, reaction with selenium powder in lieu of diorganyldiselenide provided the cyclic ebselen derivatives via C−Se/N−Se bond formation cascade. Thus, we provide here a practical and user-friendly protocol for C(sp2)-H selenation combining inexpensive first row-transition metal catalysts coupled with aerial oxygen as the terminal oxidant.

M3: an integrative framework for structure determination of molecular machines.

We present a broadly applicable, user-friendly protocol that incorporates sparse and hybrid experimental data to calculate quasi-atomic-resolution structures of molecular machines. The protocol uses the HADDOCK framework, accounts for extensive structural rearrangements both at the domain and atomic levels and accepts input from all structural and biochemical experiments whose data can be translated into interatomic distances and/or molecular shapes.