Biased coarse-grained molecular dynamics simulation approach for flexible fitting of X-ray structure into cryo electron microscopy maps

Several approaches have been introduced to interpret, in terms of high-resolution structure, low-resolution structural data as obtained from cryo-EM. As conformational changes are often observed in biological molecules, these techniques need to take into account the flexibility of proteins. Flexibility has been described in terms of movement between rigid domains and between rigid secondary structure elements, which present some limitations for studying dynamical properties. Normal mode analysis has also been used, but is limited to medium resolution data. All-atom molecular dynamics fitting techniques are more appropriate to fit structures into higher-resolution data as full protein flexibility is considered, but are cumbersome in terms of computational time. Here, we introduce a coarse-grained approach; a Go-model was used to represent biological molecules, combined with biased molecular dynamics to reproduce accurately conformational transitions. Illustrative examples on simulated data are shown. Accurate fittings can be obtained for resolution ranging from 5 to 20 Å. The approach was also tested on experimental data of Elongation Factor G and Escherichia coli RNA polymerase, where its validity is compared to previous models obtained from different techniques. This comparison demonstrates that quantitative flexible techniques, as opposed to manual docking, need to be considered to interpret low-resolution data.     

In situ cryopreservation of human embryonic stem cells in gas-permeable membrane culture cassettes for high post-thaw yield and good manufacturing practice

Current strategies for marine pollution monitoring are based on the integration of chemical and biological techniques. The sea urchin embryo-larval bioassays are among the biological methods most widely used worldwide. Cryopreservation of early embryos of sea urchins could provide a useful tool to overcome one of the main limitations of such bioassays, the availability of high quality biological material all year round. The present study aimed to determine the suitability of several permeant (dimethyl sulfoxide, Me₂SO; propylene glycol, PG; and ethylene glycol, EG) and non-permeant (trehalose, TRE; polyvinylpyrrolidone, PVP) cryoprotectant agents (CPAs) and their combination, for the cryopreservation of eggs and embryos of the sea urchin Paracentrotus lividus. On the basis of the CPAs toxicity, PG and EG, in combination with PVP, seem to be most suitable for the cryopreservation of P. lividus eggs and embryos. Several freezing procedures were also assayed. The most successful freezing regime consisted on cooling from 4 to −12 °C at 1 °C/min, holding for 2 min for seeding, cooling to −20 °C at 0.5 °C/min, and then cooling to −35 °C at 1 °C/min. Maximum normal larvae percentages of 41.5% and 68.5%, and maximum larval growth values of 42.9% and 60.5%, were obtained for frozen fertilized eggs and frozen blastulae, respectively.     

Rapid dissection of a complex phenotype through genomic-scale mapping of fitness altering genes

The understanding and engineering of complex phenotypes is a critical issue in biotechnology. Conventional approaches for engineering such phenotypes are often resource intensive, marginally effective, and unable to generate the level of biological understanding desired. Here, we report a new approach for rapidly dissecting a complex phenotype that is based upon the combination of genome-scale growth phenotype data, precisely targeted growth selections, and informatic strategies for abstracting and summarizing data onto coherent biological processes. We measured at high resolution (125 NT) and for the entire genome the effect of increased gene copy number on overall biological fitness corresponding to the expression of a complex phenotype (tolerance to 3-hydroxypropionic acid (3-HP) in Escherichia coli). Genetic level fitness data were then mapped according to various definitions of gene–gene interaction in order to generate network-level fitness data. When metabolic pathways were used to define interactions, we observed that genes within the chorismate and threonine super-pathways were disproportionately enriched throughout selections for 3-HP tolerance. Biochemical and genetic studies demonstrated that alleviation of inhibition of either of these super-pathways was sufficient to mitigate 3-HP toxicity. These data enabled the design of combinatorial modifications that almost completely offset 3-HP toxicity in minimal medium resulting in a 20 g/L and 25-fold increase in tolerance and specific growth, respectively.     

Diabetogenic action of human growth hormone. Synthesis and activity of C-terminal fragments.

Three peptides corresponding to the C-terminal region of human growth hormone have been synthesized by the solid-phase method: HGH-(177--191), HGH-(178--191) and HGH-(179--191). The diabetogenic activities of these synthetic peptides are reported. The data indicate that extension of HGH-(179--191) at its NH2-terminus is required for in vivo activity. The reduced and S-carbamidomethylated form of HGH-(177--191) was also active, indicating that the disulphide bond is possibly not a prerequisite for biological activity.     

Interactions of bovine serum albumin with biological buffers,TES, TAPS,and TAPSO in aqueous solutions

The thermal stability of bovine serum albumin (BSA) in the aqueous solutions containing the biological buffers, N-[tris(hydroxymethyl)methyl]-2-aminoethanesulfonic acid (TES), N-[tris(hydroxylmethyl)methyl]-3-aminopropanesulfonic acid (TAPS), and N-[tris(hydroxymethyl)methyl]-3-amino-2-hydroxypropanesulfonic acid (TAPSO), was studied by using dynamic light scattering (DLS) at various temperatures and concentration ranges of buffers. It is found that the increase of the buffer concentration enhanced the thermal stability of protein BSA, and the stabilization tendency follows the order of TAPSO > TES ≈ TAPS. In this study, we have also investigated the interactions of BSA with TES, TAPS, and TAPSO by using various techniques, such as UV–vis absorption, fluorescence, and molecular docking. It is revealed that the main interactions between the studied buffers and the peptide moieties of proteins are electrostatic including hydrogen bonds. The results obtained from this series of studies confirmed that the biological buffers, TES, TAPS, and TAPSO can serve as good stabilizers for the globular protein BSA, in the aqueous solutions.     

Platform design for extraction and isolation of Bromelain: Complex formation and precipitation with carrageenan

The main objective of this work was to investigate for the first time the molecular mechanism of complex formation between bromelain (a positively charged enzyme) and carrageenan (a natural strong polyelectrolyte, negatively charged) using spectroscopy techniques and thermodynamic approaches. The Bromelain-Carrageenan complex showed a maximal non-solubility at pH around 5.1. The solubility was dependent on pH and ionic strength of the medium. To re-dissolve the formed complex, the pH was changed and 500 mM of NaCl was added to the initial solution, proving the columbic mechanism for the formation of non-soluble complex. The formation of the carrageenan-bromelain complex increased in 8 °C the enzyme thermal stability, while its biological activity was not modified. The amount of total enzyme recovered in solution after precipitation with around 0.08% w/v of carrageenan was 85–90%.     

Comparability of macroinvertebrate biomonitoring indices of river health derived from semi-quantitative and quantitative methodologies

Aquatic macroinvertebrates have been the basis for one of the primary indicators and a cornerstone of lotic biomonitoring for over 40 years. Despite the widespread use of lotic invertebrates in statutory biomonitoring networks, scientific research and citizen science projects, the sampling methodologies employed frequently vary between studies. Routine statutory biomonitoring has historically relied on semi-quantitative sampling methods (timed kick sampling), while much academic research has favoured fully quantitative methods (e.g. Surber sampling). There is an untested assumption that data derived using quantitative and semi-quantitative samples are not comparable for biomonitoring purposes. As a result, data derived from the same site, but using different sampling techniques, have typically not been analysed together or directly compared. Here, we test this assumption by comparing a range of biomonitoring metrics derived from data collected using timed semi-quantitative kick samples and quantitative Surber samples from the same sites simultaneously. In total, 39 pairs of samples from 7 rivers in the UK were compared for two seasons (spring and autumn). We found a strong positive correlation (r_s = +0.84) between estimates of taxa richness based on ten Surber sub-samples and a single kick sample. The majority of biomonitoring metrics were comparable between techniques, although only fully quantitative sampling allows the density of the community (individual m⁻²) to be determined. However, this advantage needs to be balanced alongside the greater total sampling time and effort associated with the fully quantitative methodology used here. Kick samples did not provide a good estimate of relative abundance of a number of species/taxa and, therefore, the quantitative method has the potential to provide important additional information which may support the interpretation of the biological metrics.     

Inhibition kinetics of a commercial mixed culture by ammonium sulfate

The inhibitory effect of ammonium sulfate on a commercial mixed culture, used in biological waste-water treatment was studied under aerobic batch conditions. Several mathematical models of enzyme and growth kinetics including a death factor were analyzed through nonlinear regression to find the best fit to corresponding data of inhibition. The best fit model was found to be the generalized Monod type with a death factor having the biokinetic parameters; μ_max 0.681 h⁻¹, K_s 0.224 g dm⁻³, K_i 56240 g dm⁻³, K 0.055 g dm⁻³ and k_d 0.052 h⁻¹ to represent the experimental data accurately. The low saturation coefficient value along with high maximum specific growth rate and inhibition coefficient denotes the competitive characteristics of commercial mixed cultures in the biological treatment of high ammonium polluted waste waters.     

Plant species and communities assessment in interaction with edaphic and topographic factors; an ecological study of the mount Eelum District Swat,Pakistan

The current analyses of vegetation were aimed to study the different effects of environmental variables and plant species and communities interaction to these variables, identified threats to local vegetation and suggestion for remedial measures in the Mount Eelum, Swat, Pakistan. For assessment of environmental variability quantitative ecological techniques were used through quadrats having sizes of 2 × 2, 5 × 5 and 10 × 10 m² for herbs, shrubs and trees respectively. Result of the present study revealed 124 plant species in the study area. Canonical Correspondence Analysis (CCA) was used to analyze the ecological gradient of vegetation. The environmental data and species abundance were used in CANOCO software version 4.5. The presence absence data of plant species were elaborated with Cluster and Two Way Cluster Analysis techniques using PC-ORD version 5 to show different species composition that resulted in five plant communities. Findings indicate that elevation, aspect and soil texture are the strongest variables that have significant effect on species composition and distribution of various communities shown with P value 0.0500. It is recommended to protect and use sensibly whole of the Flora normally and rare species particularly in the region.     

Structural determinants of the enantioselectivity of the hydroxynitrile lyase from Hevea brasiliensis

The hydroxynitrile lyase from the tropical rubber tree Hevea brasiliensis (HbHNL) is utilized as a biocatalyst in stereospecific syntheses of alpha-hydroxynitriles from aldehydes and methyl-ketones. The catalyzed reaction represents one of the few industrially relevant examples of enzyme mediated C-C coupling reactions. In this work, we determined the X-ray crystal structures (at 1.54 and 1.76 Angstroms resolution) of HbHNL complexes with two chiral substrates -- mandelonitrile and 2,3-dimethyl-2-hydroxy-butyronitrile -- by soaking and rapid freeze quenching techniques. This is the first structural observation of the complex between a HNL and chiral substrates. Consistent with the known selectivity of the enzyme, only the S-enantiomers of the two substrates were observed in the active site. The binding modes of the chiral substrates were identical to that observed for the biological substrate acetone cyanohydrin. This indicates that the transformation of these non-natural substrates follows the same mechanism. A large hydrophobic pocket was identified in the active site of HbHNL which accommodates the more voluminous substituents of the two substrates. A three-point binding mode of the substrates -- hydrophobic pocket, hydrogen bonds between the hydroxyl group and Ser80 and Thr11, electrostatic interaction of the cyano group with Lys236 -- offers a likely structural explanation for the enantioselectivity of the enzyme. The structural data rationalize the observed (S)-enantioselectivity and form the basis for modifying the stereospecificity through rational design. The structures also revealed the necessity of considerable flexibility of the sidechain of Trp128 in order to bind and transform larger substrates.     

The inheritance of pathogenic mitochondrial DNA mutations

Mitochondrial DNA mutations cause disease in > 1 in 5000 of the population, and ～ 1 in 200 of the population are asymptomatic carriers of a pathogenic mtDNA mutation. Many patients with these pathogenic mtDNA mutations present with a progressive, disabling neurological syndrome that leads to major disability and premature death. There is currently no effective treatment for mitochondrial disorders, placing great emphasis on preventing the transmission of these diseases. An empiric approach can be used to guide genetic counseling for common mtDNA mutations, but many families transmit rare or unique molecular defects. There is therefore a pressing need to develop techniques to prevent transmission based on a solid understanding of the biological mechanisms. Several recent studies have cast new light on the genetics and cell biology of mtDNA inheritance, but these studies have also raised new controversies. Here we compare and contrast these findings and discuss their relevance for the transmission of human mtDNA diseases.     

Aqueous extract of gum olibanum (Boswellia serrata): A reductant and stabilizer for the biosynthesis of antibacterial silver nanoparticles

A simple and ecofriendly biosynthetic process has been developed for silver nanoparticles using the aqueous extract of gum olibanum (Boswellia serrata), a renewable natural plant biopolymer. The water soluble compounds in the gum serve as dual functional reducing and stabilizing agents. The effect of concentration of gum and silver nitrate; and reaction time on nanoparticle synthesis was studied. The UV–visible spectroscopy, transmission electron microscopy and X-ray diffraction techniques were used to characterize the synthesized nanoparticles. By tuning the reaction conditions, size controlled spherical nanoparticles of around 7.5 ± 3.8 nm was achieved. Using Fourier transform infrared spectroscopy and Raman spectroscopy, a probable mechanism involved in reduction and stabilization of nanoparticles has been explained. The produced silver nanoparticles exhibited substantial antibacterial activity on both the Gram classes of bacteria. By virtue of being biogenic and encapsulated with proteins, these surface functionalized nanoparticles can be easily integrated for various biological applications.     

Ventriculographie isotopique : comparaison de l’acquisition sur gamma caméra CZT cardio dédiée à l’acquisition planaire

IntroductionGated equilibrium radionuclide venticulography is often used to determine left ventricule ejection fraction (LVEF), especially in case of follow up when cardio-toxic drugs are administred. During the last decade, the use of cardiac specific (CZT) gamma cameras has spread. They directly acquire 3D data. We wanted to determine the agreement between three nuclear medicine LVEF measurement techniques. We also wanted to determine the repeatability of these techniques.MethodsBetween April 16, 2016 and February 4, 2017 we consecutively included 77 patients who were adressed to the nuclear medicine department of CHU Martinique for LVEF measurement. Patients were injected with 99*Technetium labelled albumin. Usual planar scintigraphy was performed to serve as reference and 3D data was acquired with a CZT cardiac gamma camera. 3D data was analyzed directly with BPGS software, and also reprojected to planar data which was analyzed like the conventional planar acquisition.ResultsSeventy patients had acquisitions with both gamma cameras. The LVEF values from the CZT camera data were significantly higher than the reference planar LVEF measurements (+6.2 for 3D analysis, P < 0.01, concordance interval [?8.0; 20.3] and +1.7 for planar reprojection, P = 0.01, concordance interval [?8.9; 12.2]. For all three methods, the intra-method concordance intervals were within [?5; 5].ConclusionOur results indicate the same measurement method must be used in case of LVEF follow up. The variation between theses methods could lead to the false conclusion of impairment of LVEF (which is suspected in case of a 10% LVEF drop). Our secondary outcome show a good repeatability for all three techniques. Further studies should be initiated to determine which method is the most accurate and to determine the reproductibility of the CZT data acquisition process.     

Alginate embedding and subsequent sporulation of in vitro-produced Conidiobolus thromboides hyphae using a pressurised air-extrusion method

Conidiobolus thromboides is an entomophthoralean fungus with potential as a biological control agent of aphids. However, its application in biological control is limited due to its formulation requirements. The objective of this study was to develop and optimise a novel air-extrusion method to embed C. thromboides hyphae at high density in alginate pellets. An orthogonal experimental design was used to investigate selected combinations of parameters known to affect hyphal density within pellets. The diameter of pellets produced, and the calculated density of hyphae within them, ranged from 0.18 ± 0.09 to 3.17 ± 0.06 mm and from 0.02 to 350.56 mg/mm³ respectively. These data were used to predict the optimal parameter combination to deliver the greatest density of hyphae of C. thromboides per pellet: 1% sodium alginate, a 1:2 ratio of hyphae to sodium alginate, an orifice diameter of 0.232 mm and an air pressure of 0.05 MPa. Pellets made under the optimal conditions predicted produced a mean total of 4.3 ± 0.6 × 10⁵ conidia per pellet at 100% relative humidity which was significantly greater than the mean total number of conidia produced from infected aphid cadavers of comparable size (9.35 ± 0.85 × 10⁴) (p < 0.001). In conclusion, air-extrusion embedding appears to be a promising method for formulating in vitro-produced hyphae of C. thromboides for use in biological control.     

A self-evaluation method to attribute sound bio-values based on in situ ecotoxicological monitoring

Microbial, plant and animal species were examined within the framework of the ADEME Bioindicator Program in France that involves the monitoring, characterization and risk assessment of soils from various sites. Whenever any of the selected species under study were not found in all of the sampling areas of a given site, the data were difficult to analyze. It is explained how discrepancies in the data analysis may arise depending on the way the values of the biological parameters are taken into account: as a biomarker to assess values for each species in different areas, or as bioindicator to evaluate the number of species impacted in each area. We present a method to prevent discrepancies in interpretation in which a bio-value (“soil quality”) is attributed to various areas, allowing them to be ranked. The method also provides an evaluation of the soundness of the results and the resulting ranking, and no areas need to be assigned a priori as “control” or “contaminated” areas.In the framework of the ADEME Bioindicator Program, the leaf C18:3/(C18:0 + C18:1 + C18:2) fatty acid ratio was used as biological parameter. The experimental data obtained from two sites, in which not all plant species under study were found in all sampling areas, were analyzed by the above-mentioned method. The method proved usable to calculate a sound bio-value for each area in both sites and to rank them. Comparison of data demonstrated lower bio-values for the soil in the four areas within the industrial wasteland than for that in the two areas located just outside it. The soundness of the scoring obtained in the second site demonstrates that coherent results could also be obtained with plant species harvested in areas several kilometers apart and displaying varying soil properties.     

Statistical Series: Opportunities and challenges of sperm motility subpopulation analysis

Computer-assisted sperm analysis (CASA) allows assessing the motility of individual spermatozoa, generating huge datasets. These datasets can be analyzed using data mining techniques such as cluster analysis, to group the spermatozoa in subpopulations with biological meaning. This review considers the use of statistical techniques for clustering CASA data, their challenges and possibilities. There are many clustering approaches potentially useful for grouping sperm motility data, but some options may be more appropriate than others. Future development should focus not only in improvements of subpopulation analysis, but also in finding consistent biological meanings for these subpopulations.     

Two novel methods to assess ulnar nerve conduction across the elbow

PurposeNerve conduction studies (NCS) are used as an electrodiagnostic method for diagnosing ulnar neuropathy of the elbow (UNE). The purpose of this study was to determine normal and reliability values of across elbow ulnar nerve conduction velocity using two novel methods.MethodsUlnar nerve conduction studies were performed on both upper extremities of 104 healthy subjects. Two different techniques were used to evaluate ulnar nerve function at the elbow: Technique 1 (W-BE-AE) determined mixed NCV across the elbow indirectly while Technique 2 (BE-AE) measured conduction time directly. Twenty subjects returned within one week for re-testing to generate reliability data.ResultsThe mean NCV for the BE-AE segment using Technique 1 was 59.68 m/s (±8.91 m/s). The mean peak latency for the BE-AE segment using Technique 2 was 2.03 ms (±0.24 ms). The interrater and intrarater reliability intraclass correlation coefficient (ICC) for Technique 1 was 0.454 and 0.756, respectively. For Technique 2, the interrater and intrarater reliability ICC was 0.76 and 0.814, respectively.ConclusionThis study identified normal values for ulnar nerve conduction across the elbow with reliability ranging from poor to good, depending on the technique. These two novel techniques provide alternative methods to traditional techniques to measure ulnar nerve conduction across the elbow.     

黄瓜花叶病毒卫星RNA生防制剂在烟草上的防病作用

用人工组装的黄瓜花叶病毒卫星RNA生防制剂在烟草生产品种进行了田间保护试验。由于使用了生防制剂,经处理的烟草发病率降低84.9～86.4％,同时比对照早熟5天,上等烟比率增加151.3～173.5％,平均每亩收入增加43.3～65.7％。另外发现生防制剂能增强烟草对真菌病害的抵抗作用。     

Classification in conservation biology: A comparison of five machine-learning methods

Classification is one of the most widely applied tasks in ecology. Ecologists have to deal with noisy, high-dimensional data that often are non-linear and do not meet the assumptions of conventional statistical procedures. To overcome this problem, machine-learning methods have been adopted as ecological classification methods. We compared five machine-learning based classification techniques (classification trees, random forests, artificial neural networks, support vector machines, and automatically induced rule-based fuzzy models) in a biological conservation context. The study case was that of the ocellated turkey (Meleagris ocellata), a bird endemic to the Yucatan peninsula that has suffered considerable decreases in local abundance and distributional area during the last few decades. On a grid of 10 × 10 km cells that was superimposed to the peninsula we analysed relationships between environmental and social explanatory variables and ocellated turkey abundance changes between 1980 and 2000. Abundance was expressed in three (decrease, no change, and increase) and 14 more detailed abundance change classes, respectively. Modelling performance varied considerably between methods with random forests and classification trees being the most efficient ones as measured by overall classification error and the normalised mutual information index. Artificial neural networks yielded the worst results along with linear discriminant analysis, which was included as a conventional statistical approach. We not only evaluated classification accuracy but also characteristics such as time effort, classifier comprehensibility and method intricacy—aspects that determine the success of a classification technique among ecologists and conservation biologists as well as for the communication with managers and decision makers. We recommend the combined use of classification trees and random forests due to the easy interpretability of classifiers and the high comprehensibility of the method.