Determination of trimethoprim in bovine serum by high-performance liquid chromatography with confirmation by thermospray liquid chromatography—mass spectrometry

A high-performance liquid chromatographic (HPLC) method with a detection limit of 5 ng/ml was developed for the analysis of trimethoprim in bovine serum. Trimethoprim and the internal standard, ormetoprim, under alkaline conditions, were first extracted into dichloromethane and then back-extracted into dilute sulphuric acid (0.15 M) and cleaned-up on a C₁₈ cartridge. Trimethoprim was quantified on a C₁₈ column using a triethylammonium acetate—acetonitrile—methanol (16:3:1, v/v/v) mobile phase at a flow-rate of 1.5 ml/min, with ultraviolet detection at 225 nm. This method was used to verify the accuracy of test responses obtained with the Brilliant Black Reduction test, a rapid screening method, for trimethoprim levels in the serum of steers treated with Trivetrin. Confirmation of the presence of trimethoprim in the sample extract was obtained by thermospray HPLC—mass spectrometry.     

Design and preclinical testing of an anti-CD41 CAR T cell for the treatment of acute megakaryoblastic leukaemia

Acute megakaryoblastic leukaemia (AMkL) is a rare subtype of acute myeloid leukaemia (AML) representing 5% of all reported cases, and frequently diagnosed in children with Down syndrome. Patients diagnosed with AMkL have low overall survival and have poor outcome to treatment, thus novel therapies such as CAR T cell therapy could represent an alternative in treating AMkL. We investigated the effect of a new CAR T cell which targets CD41, a specific surface antigen for M7-AMkL, against an in vitro model for AMkL, DAMI Luc2 cell line. The performed flow cytometry evaluation highlighted a percentage of 93.8% CAR T cells eGFP-positive and a limited acute effect on lowering the target cell population. However, the interaction between effector and target (E:T) cells, at a low ratio, lowered the cell membrane integrity, and reduced the M7-AMkL cell population after 24 h of co-culture, while the cytotoxic effect was not significant in groups with higher E:T ratio. Our findings suggest that the anti-CD41 CAR T cells are efficient for a limited time spawn and the cytotoxic effect is visible in all experimental groups with low E:T ratio.     

RosettaDDGPrediction for high-throughput mutational scans: From stability to binding

Reliable prediction of free energy changes upon amino acid substitutions (ΔΔGs) is crucial to investigate their impact on protein stability and protein–protein interaction. Advances in experimental mutational scans allow high-throughput studies thanks to multiplex techniques. On the other hand, genomics initiatives provide a large amount of data on disease-related variants that can benefit from analyses with structure-based methods. Therefore, the computational field should keep the same pace and provide new tools for fast and accurate high-throughput ΔΔG calculations. In this context, the Rosetta modeling suite implements effective approaches to predict folding/unfolding ΔΔGs in a protein monomer upon amino acid substitutions and calculate the changes in binding free energy in protein complexes. However, their application can be challenging to users without extensive experience with Rosetta. Furthermore, Rosetta protocols for ΔΔG prediction are designed considering one variant at a time, making the setup of high-throughput screenings cumbersome. For these reasons, we devised RosettaDDGPrediction, a customizable Python wrapper designed to run free energy calculations on a set of amino acid substitutions using Rosetta protocols with little intervention from the user. Moreover, RosettaDDGPrediction assists with checking completed runs and aggregates raw data for multiple variants, as well as generates publication-ready graphics. We showed the potential of the tool in four case studies, including variants of uncertain significance in childhood cancer, proteins with known experimental unfolding ΔΔGs values, interactions between target proteins and disordered motifs, and phosphomimetics. RosettaDDGPrediction is available, free of charge and under GNU General Public License v3.0, at https://github.com/ELELAB/RosettaDDGPrediction .     

Bioactivity and molecular modelling of diphenylsulfides and diphenylselenides

Bis(2-bromo-4,5-dimethoxyphenyl)sulfide (5) and bis(2-bromo-4,5-dimethoxyphenyl) selenide (7) have been shown to block cells in the G2/M phase of the cell cycle, whereas the debromo (4, 6) equivalents do not. The dibromoselenide (7) is cytotoxic to tumour cells in vitro and has been shown to increase the mitotic index of treated cells. These biological effects are consistent with disruption of the mitotic apparatus. This agent does not inhibit microtubule assembly in vitro, but does bind to tubulin. Molecular modelling of these structures indicates that their spatial and electronic structures may make an important contribution to the biological activity.     

The Ontogeny of Vocal Sequences: Insights from a Newborn Wild Chimpanzee (Pan troglodytes schweinfurthii)

International Journal of Primatology - Observations of early vocal behaviours in non-human primates (hereafter primates) are important for direct comparisons between human and primate vocal...     

Correlation between protein stability and crystal properties of designed ROP variants

Six variants of the ROP protein, designed with the aim to analyze by X-ray crystallography loop formation and core packing interactions in 4-α-helical bundles- have been purified and a search of their crystallization conditions has been carried out. Five mutants yield crystals that are suitable for medium to high resolutionX-ray diffraction studies. For all mutants crystal size- sensitivity to X-irradiation and diffraction limit are correlated to their stability as determined by differential scanning calorimetry- in a manner which is not yet understood in detail. © Wiley-Liss, Inc.     

A chromosome-level genome assembly enables the identification of the follicule stimulating hormone receptor as the master sex-determining gene in the flatfish Solea senegalensis

Sex determination (SD) shows huge variation among fish and a high evolutionary rate, as illustrated by the Pleuronectiformes (flatfishes). This order is characterized by its adaptation to demersal life, compact genomes and diversity of SD mechanisms. Here, we assembled the Solea senegalensis genome, a flatfish of great commercial value, into 82 contigs (614 Mb) combining long- and short-read sequencing, which were next scaffolded using a highly dense genetic map (28,838 markers, 21 linkage groups), representing 98.9% of the assembly. Further, we established the correspondence between the assembly and the 21 chromosomes by using BAC-FISH. Whole genome resequencing of six males and six females enabled the identification of 41 single nucleotide polymorphism variants in the follicle stimulating hormone receptor (fshr) consistent with an XX/XY SD system. The observed sex association was validated in a broader independent sample, providing a novel molecular sexing tool. The fshr gene displayed differential expression between male and female gonads from 86 days post-fertilization, when the gonad is still an undifferentiated primordium, concomitant with the activation of amh and cyp19a1a, testis and ovary marker genes, respectively, in males and females. The Y-linked fshr allele, which included 24 nonsynonymous variants and showed a highly divergent 3D protein structure, was overexpressed in males compared to the X-linked allele at all stages of gonadal differentiation. We hypothesize a mechanism hampering the action of the follicle stimulating hormone driving the undifferentiated gonad toward testis.     

FACTORS AFFECTING RED SPRUCE REGENERATION IN DECLINING AREAS OF CAMELS HUMP MOUNTAIN,VERMONT

Scarcity of red spruce (Picea rubens Sarg.) seedlings in declining spruce-fir forests of Camels Hump mountain, Vermont, prompted a study on some contributing factors involved in failure of spruce regeneration. Cones were shorter than those from low elevation red spruce trees from unaffected sites. Seed number in cones collected in declining areas of Camels Hump was low as were seed sizes and weights. Seed germination was at control levels only in good seed years. Capacity of seeds to form seedlings was reduced relative to that of controls, although seedling growth was normal. Coniferous litter contains presumed allelopathic substances leachable by contemporary precipitations that affect seed germination and seedling root development in red spruce, but not in balsam fir. Shield fern contains leachable substances that reduce seed germination and seedling root development in red spruce, but not balsam fir. Roots of red spruce germlings have lower capacity to penetrate through the increased forest duff depths of declining forests than do balsam fir roots. It is anticipated that substantial reproduction of red spruce will not occur in declining montane conifer forests under present conditions.     

North Sea cod and climate change – modelling the effects of temperature on population dynamics

In order to examine the likely impacts of climate change on fish stocks, it is necessary to couple the output from large‐scale climate models to fisheries population simulations. Using projections of future North Sea surface temperatures for the period 2000–2050 from the Hadley General Circulation Model, we estimate the likely effects of climate change on the North Sea cod population. Output from the model suggests that increasing temperatures will lead to an increased rate of decline in the North Sea cod population compared with simulations that ignore environmental change. Although the simulation developed here is relatively simplistic, we demonstrate that inclusion of environmental factors in population models can markedly alter one's perception of how the population will behave. The development of simulations incorporating environment effects will become increasingly important as the impacts of climate change on the marine ecosystem become more pronounced.     

Uptake of bacteriophage λ DNA in Escherichia coli by electroporation: An alternative to in vitro packaging

Summary By using a high field strength DC pulse of 15 kV/cm and a pulse duration of 5 ms for the transfection of E. coli by bacteriophage DNA, we obtained efficiencies of 1.1 × 10⁶ (pfu/g bacteriophage , DNA). This represents a 100-fold improvement over the traditional CaCl₂/heat shock method and is a viable alternative to the more costly in vitro packaging of recombinant bacteriophage DNA for the production of cDNA and genomic libraries.