Film-like organelles equip cells with multiple genetic codes.

The simultaneous incorporation of distinct noncanonical amino acids into different proteins within eukaryotic cells remains challenging. This new study by Reinkemeierand Lemke demonstrates how 2D phase separation can be used to engineer spatially separated organelles. These film-like organelles translate proteins independently from each other and the canonical genetic code.

Evaluating Nanoparticle Hydrophobicity Using Analytical Membrane Hydrophobic Interaction Chromatography.

Nanoparticle hydrophobicity is a key factor controlling the stability, adhesion, and transport of nanoparticle suspensions. Although a number of approaches have been presented for evaluating nanoparticle hydrophobicity, these methods are difficult to apply to larger nanoparticles and viruses (>100 nm in size) that are of increasing importance in drug delivery and gene therapy. This study investigated the use of a new analytical hydrophobic interaction chromatography method employing a 5.0 µm pore size polyvinylidene fluoride membrane as the stationary-phase in membrane hydrophobic interaction chromatography (MHIC). Experimental data obtained using a series of model proteins were in good agreement with literature values for the hydrophobicity (both experimental and computational). MHIC was then used to evaluate the hydrophobicity of a variety of nanoparticles, including a live attenuated viral vaccine, both in water and in the presence of different surfactants. This new method can be implemented on any liquid chromatography system, run times are typically <20 min, and the experiments avoid the use of organic solvents that could alter the structure of many biological nanoparticles.

Remote classroom modelling: a professional development model for in-service generalist primary teachers of science.

We present a model for Professional Development (PD) for in-service generalist primary teachers of science. The Remote Classroom Modelling (RCM) model is specifically designed to address salient challenges in the context of professional isolation. We share the principles that supported the design of this PD prototype, and the insights and lessons learned from a pilot of the model. We employed open-ended questionnaires, individual interviews and focus group interviews using a qualitative exploratory study to capture the experiences of teachers and students involved in this trial. Our findings suggest that the model supports primary teachers in developing: (1) their technological, pedagogical, and content knowledge, (2) their leadership, and (3) their resourcefulness in science. Our analysis reveals two important factors underpinning the successful implementation of this model: participant engagement and expert support. We discuss the applicability of this model in different settings and propose an agenda to progress the development of the RCM.

Evaluation of a sterile filtration process for viral vaccines using a model nanoparticle suspension.

There is growing interest in the development of new vaccines based on live-attenuated viruses (LAVs) and virus-like particles. The large size of these vaccines, typically 100-400 nm, significantly complicates the use of sterile filtration. The objectives of this study are to examine the performance of several commercial sterile filters for filtration of a cytomegalovirus vaccine candidate (referred to as the LAV) and to develop and evaluate the use of a model nanoparticle suspension to perform a more quantitative assessment. Data obtained with a mixture of 200- and 300-nm fluorescent particles provided yield and pressure profiles that captured the behavior of the viral vaccine. This included the excellent performance of the Sartorius Sartobran P filter, which provided greater than 80% yield of both the vaccine and model particles even though the average particle size was more than 250 nm. The particle yield for the Sartobran P was independent of filtrate flux above 200 L/m2 /h, but increased with increasing particle concentration, varying from less than 10% at concentrations around 107 particles/ml to more than 80% at concentrations above 1010 particles/ml due to saturation of particle capture/binding sites within the filter. These results provide important insights into the factors controlling transmission and fouling during sterile filtration of large vaccine products.

Investigating eosin Y as a photocatalyst for the radical-dependent activity-based probing of deubiquitinating enzymes.

Eosin Y was assessed for its ability to induce a thiol-ene dependent protein-protein reaction in a metal-free, oxygen-tolerant, visible light mediated system. Protein-protein coupling efficiency under these mild conditions was comparable to previously reported UV-dependent conditions. The desired thiol-ene reaction was however limited within more complex biological systems.

Omani senior secondary school students' knowledge of and attitudes to antibiotic resistance.

Antibiotic resistance is a worldwide problem that is increasing largely due to the misuse of antibiotics in human health and agriculture. This situation is further exacerbated by a dearth of new antibiotic development, the focus of pharmaceutical companies having shifted to more lucrative treatments for chronic conditions such as elevated blood pressure. To conserve the efficacy of the current crop of antibiotics, it is vital that they are used appropriately by individuals. Effective education may be a means to achieve such appropriate use. This paper reports on a large-scale, mixed methods study, which employed a survey and oral questionnaires, undertaken with senior secondary Omani students. The study explored students' understanding of antibiotic resistance as well as their attitudes to the issue of antibiotic resistance. The study findings indicated that, although some students had a reasonably clear understanding of antibiotic resistance, many had serious misconceptions that could result in misuse of antibiotics. The article concludes with suggestions for amending secondary school pedagogy in Oman to address the misconceptions.

Bacterial Retention During Filtration of a Live Attenuated Virus Vaccine Through the Sartobran P Sterile Filter.

Recent studies of sterile filtration of a Live Attenuated Virus (LAV) demonstrated that the Sartobran P sterile filter provided 80% yield of a LAV that was 100 - 400 nm in size, raising questions about the effectiveness of this filter in retaining the standard challenge bacterium, Brevundimonas diminuta. This study evaluated the retention of B. diminuta by the Sartobran P over a range of conditions appropriate for LAV filtration. The B. diminuta were characterized by dynamic light scattering (DLS), nanoparticle tracking analysis (NTA), and scanning electron microscopy. The Sartobran P showed complete retention of B. diminuta under all conditions, even in the presence of additives like sucrose, surfactants, and high salt that have previously been hypothesized to increase the risk of bacterial breakthrough. The size of B. diminuta decreased when incubated in the nutrient poor media required by the ASTM challenge test. The addition of sucrose caused a further reduction in size as measured by NTA, although this was due to an increase in cell motility. There was no evidence of bacterial breakthrough at high loadings of either the LAV or B. diminuta, further demonstrating the effectiveness of the Sartobran P for sterile filtration of large viral vaccines.

A role for Arabidopsis dynamin related proteins DRP2A/B in endocytosis; DRP2 function is essential for plant growth.

Endocytosis is an essential cellular process that allows cells to internalise proteins and lipid from the plasma membrane to change its composition and sense and respond to alterations in their extracellular environment. In animal cells, the protein dynamin is involved in membrane scission during endocytosis, allowing invaginating vesicles to become internalised. Arabidopsis encodes two proteins that have all the domains essential for function in the animal dynamins, Dynamin Related Proteins 2A and 2B (DRP2A and 2B). These proteins show very high sequence identity and are both expressed throughout the plant. Single mutants exhibited no obvious phenotypes but double mutants could be recovered as gametophytes carrying mutant copies of both DRP2A and DRP2B were not transmitted to the next generation. Immunolabelling localised DRP2A/B to the tips of root hairs, a site where rapid endocytosis takes place. Constitutive expression of a GTPase defective Dominant Negative form of DRP2A/B did not allow the recovery of plants expressing this protein at a detectable level, demonstrating an interference with endogenous dynamin. Using an inducible expression system Dominant Negative protein was transiently expressed at levels several fold that of the endogenous proteins. Inducible expression of the Dominant Negative protein resulted in reduced endocytosis at the tips of root hairs, as measured by internalisation of an endocytic tracer dye, and resulted in root hairs bulging and bursting. Together these data support a role for DRP2A/B in endocytosis in Arabidopsis, and demonstrates that the function of at least one of these closely related proteins is essential for plant growth.

Rationalizing tight ligand binding through cooperative interaction networks.

Small modifications of the molecular structure of a ligand sometimes cause strong gains in binding affinity to a protein target, rendering a weakly active chemical series suddenly attractive for further optimization. Our goal in this study is to better rationalize and predict the occurrence of such interaction hot-spots in receptor binding sites. To this end, we introduce two new concepts into the computational description of molecular recognition. First, we take a broader view of noncovalent interactions and describe protein-ligand binding with a comprehensive set of favorable and unfavorable contact types, including for example halogen bonding and orthogonal multipolar interactions. Second, we go beyond the commonly used pairwise additive treatment of atomic interactions and use a small world network approach to describe how interactions are modulated by their environment. This approach allows us to capture local cooperativity effects and considerably improves the performance of a newly derived empirical scoring function, ScorpionScore. More importantly, however, we demonstrate how an intuitive visualization of key intermolecular interactions, interaction networks, and binding hot-spots supports the identification and rationalization of tight ligand binding.

Thiol isomerases negatively regulate the cellular shedding activity of ADAM17.

ADAM17 (where ADAM is 'a disintegrin and metalloproteinase') can rapidly modulate cell-surface signalling events by the proteolytic release of soluble forms of proligands for cellular receptors. Many regulatory pathways affect the ADAM17 sheddase activity, but the mechanisms for the activation are still not clear. We have utilized a cell-based ADAM17 assay to show that thiol isomerases, specifically PDI (protein disulfide isomerase), could be responsible for maintaining ADAM17 in an inactive form. Down-regulation of thiol isomerases, by changes in the redox environment (for instance as elicited by phorbol ester modulation of mitochondrial reactive oxygen species) markedly enhanced ADAM17 activation. On the basis of ELISA binding studies with novel fragment antibodies against ADAM17 we propose that isomerization of the disulfide bonds in ADAM17, and the subsequent conformational changes, form the basis for the modulation of ADAM17 activity. The shuffling of disulfide bond patterns in ADAMs has been suggested by a number of recent adamalysin crystal structures, with distinct disulfide bond patterns altering the relative orientations of the domains. Such a mechanism is rapid and reversible, and the role of thiol isomerases should be investigated further as a potential factor in the redox regulation of ADAM17.

Role of furin in granular acidification in the endocrine pancreas: identification of the V-ATPase subunit Ac45 as a candidate substrate.

Furin is a proprotein convertase which activates a variety of regulatory proteins in the constitutive exocytic and endocytic pathway. The effect of genetic ablation of fur was studied in the endocrine pancreas to define its physiological function in the regulated secretory pathway. Pdx1-Cre/loxP furin KO mice show decreased secretion of insulin and impaired processing of known PC2 substrates like proPC2 and proinsulin II. Both secretion and PC2 activity depend on granule acidification, which was demonstrated to be significantly decreased in furin-deficient beta cells by using the acidotrophic agent 3-(2,4-dinitroanilino)-3'amino-N-methyldipropylamine (DAMP). Ac45, an accessory subunit of the proton pump V-ATPase, was investigated as a candidate substrate. Ac45 is highly expressed in islets of Langerhans and furin was able to cleave Ac45 ex vivo. Furthermore, the exact cleavage site was determined. In addition, reduced regulated secretion and proinsulin II processing could be obtained in the insulinoma cell line betaTC3 by downregulation of either furin or Ac45. Together, these data establish an important role for furin in regulated secretion, particularly in intragranular acidification most likely due to impaired processing of Ac45.

Determination of X-ray detection limit and applications in perovskite X-ray detectors.

X-ray detection limit and sensitivity are important figure of merits for perovskite X-ray detectors, but literatures lack a valid mathematic expression for determining the lower limit of detection for a perovskite X-ray detector. In this work, we present a thorough analysis and new method for X-ray detection limit determination based on a statistical model that correlates the dark current and the X-ray induced photocurrent with the detection limit. The detection limit can be calculated through the measurement of dark current and sensitivity with an easy-to-follow practice. Alternatively, the detection limit may also be obtained by the measurement of dark current and photocurrent when repeatedly lowering the X-ray dose rate. While the material quality is critical, we show that the device architecture and working mode also have a significant influence on the sensitivity and the detection limit. Our work establishes a fair comparison metrics for material and detector development.

Purification of Cas9-RNA complexes by ultrafiltration.

The discovery of CRISPR-Cas9 has revolutionized molecular biology, greatly accelerating the introduction of genetic modifications into organisms and facilitating the development of novel therapeutics and diagnostics. For many applications, guide RNA and Cas9 protein are expressed, combined, and purified to produce a ribonucleic enzyme complex that is then added into a diagnostic device or delivered into cells. The objective of this work was to develop an ultrafiltration process for the selective purification of Cas9 ribonucleoprotein by removal of excess guide RNA. A His-tagged Streptococcus pyogenes Cas9 protein was produced in Escherichia coli, purified by metal affinity chromatography, and complexed with a 40 kDa (124 nucleotide) single guide RNA. Ultrafiltration experiments were first performed on solutions containing either guide RNA or Cas9 protein to identify the effect of filtration conditions and membrane pore size on the selectivity. Shear-induced aggregation of the Cas9 led to significant fouling under some conditions. A diafiltration process was then developed using a Biomax® 300 kDa polyethersulfone membrane to selectively remove excess guide RNA from a solution containing Cas9-bound guide RNA and free guide RNA. These results demonstrate the potential of using ultrafiltration for the removal of excess RNA during the production of functional ribonucleoprotein complexes.

Probing enzymatic activity - a radical approach.

Deubiquitinating enzymes (DUBs) are known to have numerous important interactions with the ubiquitin cascade and their dysregulation is associated with several diseases, including cancer and neurodegeneration. They are an important class of enzyme, and activity-based probes have been developed as an effective strategy to study them. Existing activity-based probes that target the active site of these enzymes work via nucleophilic mechanisms. We present the development of latent ubiquitin-based probes that target DUBs via a site selective, photoinitiated radical mechanism. This approach differs from existing photocrosslinking probes as it requires a free active site cysteine. In contrast to existing cysteine reactive probes, control over the timing of the enzyme-probe reaction is possible as the alkene warhead is completely inert under ambient conditions, even upon probe binding. The probe's reactivity has been demonstrated against recombinant DUBs and to capture endogenous DUB activity in cell lysate. This allows more finely resolved investigations of DUBs.

Strategies to Target Specific Components of the Ubiquitin Conjugation/Deconjugation Machinery.

The regulation of ubiquitination status in the cell is controlled by ubiquitin ligases acting in tandem with deubiquitinating enzymes. Ubiquitination controls many key processes in the cell from division to death making its tight regulation key to optimal cell function. Activity based protein profiling has emerged as a powerful technique to study these important enzymes. With around 100 deubiquitinating enzymes and 600 ubiquitin ligases in the human genome targeting a subclass of these enzymes or even a single enzyme is a compelling strategy to unpick this complex system. In this review we will discuss different approaches adopted, including activity-based probes centered around ubiquitin-protein, ubiquitin-peptide and mutated ubiquitin scaffolds. We examine challenges faced and opportunities presented to increase specificity in activity-based protein profiling of the ubiquitin conjugation/deconjugation machinery.

Cellulose biosynthesis and deposition in higher plants.

The plant cell wall is central to plant development. Cellulose is a major component of plant cell walls, and is the world's most abundant biopolymer. Cellulose contains apparently simple linear chains of glucose residues, but these chains aggregate to form immensely strong microfibrils. It is the physical properties of these microfibrils that, when laid down in an organized manner, are responsible for both oriented cell elongation during plant growth and the strength required to maintain an upright growth habit. Despite the importance of cellulose, only recently have we started to unravel details of its synthesis. Mutational analysis has allowed us to identify some of the proteins involved in its synthesis at the plasma membrane, and to define a set of cellulose synthase enzymes essential for cellulose synthesis. These proteins are organized into a very large plasma membrane-localized protein complex. The way in which this protein complex is regulated and directed is central in depositing cellulose microfibrils in the wall in the correct orientation, which is essential for directional cell growth. Recent developments have given us clues as to how cellulose synthesis and deposition is regulated, an understanding of which is essential if we are to manipulate cell wall composition.

Movement Demands of Elite U20 International Rugby Union Players.

The purpose of this study was to quantify movement demands of elite international age grade (U20) rugby union players during competitive tournament match play. Forty elite professional players from an U20 international performance squad were monitored using 10 Hz global positioning systems (GPS) during 15 international tournament matches during the 2014/15 and 2015/16 seasons. Data on distances, velocities, accelerations, decelerations, high metabolic load (HML) distance and efforts, and number of sprints were derived. Data files from players who played over 60 min (n = 161) were separated firstly into Forwards and Backs, and more specifically into six positional groups; FR--Front Row (prop & hooker), SR--Second Row, BR--Back Row (Flankers & No.8), HB--Half Backs (scrum half & outside half), MF--Midfield (centres), B3--Back Three (wings & full back) for match analysis. Analysis revealed significant differences between forwards and backs positions. Backs scored higher on all variables measured with the exception of number of moderate accelerations, decelerations (no difference). The centres covered the greatest total distance with the front row covering the least (6.51 ± 0.71 vs 4.97 ± 0.75 km, p < 0.001). The front row also covered the least high speed running (HSR) distance compared to the back three (211.6 ± 112.7 vs 728.4 ± 150.2 m, p < 0.001) who covered the most HSR distance, affirming that backs cover greater distances but forwards have greater contact loads. These findings highlight for the first time differences in the movement characteristics of elite age grade rugby union players specific to positional roles.

Interactive and Versatile Navigation of Structural Databases.

We present CSD-CrossMiner, a novel tool for pharmacophore-based searches in crystal structure databases. Intuitive pharmacophore queries describing, among others, protein-ligand interaction patterns, ligand scaffolds, or protein environments can be built and modified interactively. Matching crystal structures are overlaid onto the query and visualized as soon as they are available, enabling the researcher to quickly modify a hypothesis on the fly. We exemplify the utility of the approach by showing applications relevant to real-world drug discovery projects, including the identification of novel fragments for a specific protein environment or scaffold hopping. The ability to concurrently search protein-ligand binding sites extracted from the Protein Data Bank (PDB) and small organic molecules from the Cambridge Structural Database (CSD) using the same pharmacophore query further emphasizes the flexibility of CSD-CrossMiner. We believe that CSD-CrossMiner closes an important gap in mining structural data and will allow users to extract more value from the growing number of available crystal structures.

Movement Demands of Elite Under-20s and Senior International Rugby Union Players.

This study compared the movement demands of elite international Under-20 age grade (U20s) and senior international rugby union players during competitive tournament match play. Forty elite professional players from an U20 and 27 elite professional senior players from international performance squads were monitored using 10Hz global positioning systems (GPS) during 15 (U20s) and 8 (senior) international tournament matches during the 2014 and 2015 seasons. Data on distances, velocities, accelerations, decelerations, high metabolic load (HML) distance and efforts, and number of sprints were derived. Data files from players who played over 60 min (n = 258) were separated firstly into Forwards and Backs, and more specifically into six positional groups; FR-Front Row (prop & hooker), SR-Second Row, BR-Back Row (Flankers & No.8), HB-Half Backs (scrum half & outside half), MF-Midfield (centres), B3 -Back Three (wings & full back) for match analysis. Linear mixed models revealed significant differences between U20 and senior teams in both the forwards and backs. In the forwards the seniors covered greater HML distance (736.4 ± 280.3 vs 701.3 ± 198.7m, p = 0.01) and severe decelerations (2.38 ± 2.2 vs 2.28 ± 1.65, p = 0.05) compared to the U20s, but performed less relative HSR (3.1 ± 1.6 vs 3.2 ± 1.5, p < 0.01), moderate (19.4 ± 10.5 vs 23.6 ± 10.5, p = 0.01) and high accelerations (2.2 ± 1.9 vs 4.3 ± 2.7, p < 0.01) and sprint•min-1 (0.11 ± 0.06 vs 0.11 ± 0.05, p < 0.01). Senior backs covered a greater relative distance (73.3 ± 8.1 vs 69.1 ± 7.6 m•min-1, p < 0.01), greater High Metabolic Load (HML) distance (1138.0 ± 233.5 vs 1060.4 ± 218.1m, p < 0.01), HML efforts (112.7 ± 22.2 vs 98.8 ± 21.7, p < 0.01) and heavy decelerations (9.9 ± 4.3 vs 9.5 ± 4.4, p = 0.04) than the U20s backs. However, the U20s backs performed more relative HSR (7.3 ± 2.1 vs 7.2 ± 2.1, p <0.01) and sprint•min-1 (0.26 ± 0.07 vs 0.25 ± 0.07, p < 0.01). Further investigation highlighted differences between the 6 positional groups of the teams. The positional groups that differed the most on the variables measured were the FR and MF groups, with the U20s FR having higher outputs on HSR, moderate & high accelerations, moderate, high & severe decelerations, HML distance, HML efforts, and sprints•min-1. For the MF group the senior players produced greater values for relative distance covered, HSR, moderate decelerations, HML distance and sprint•min-1. The BR position group was most similar with the only differences seen on heavy accelerations (U20s higher) and moderate decelerations (seniors higher). Findings demonstrate that U20s internationals appear to be an adequate 'stepping stone' for preparing players for movement characteristics found senior International rugby, however, the current study highlight for the first time that certain positional groups may require more time to be able to match the movement demands required at a higher playing level than others. Conditioning staff must also bear in mind that the U20s players whilst maintaining or improving match movement capabilities may require to gain substantial mass in some positions to match their senior counterparts.

A robust clustering method for chemical structures.

A clustering method based on finding the largest set of disconnected fragments that two chemical compounds have in common is shown to be able to group structures in a way that is ideally suited to medicinal chemistry programs. We describe how markedly improved results can be obtained by using a similarity metric that accounts not just for the size of the shared fragments but also on their relative arrangement in the two parent compounds. The use of a physiochemical atom typing scheme is also shown to provide significant contributions. Results from calculations using a test set consisting of actives from nine different important biological target proteins demonstrate the strengths of our clustering method and the advantages over other approaches that are widely used throughout the pharmaceutical industry.