Directed evolution of Rhodotorula gracilisd-amino acid oxidase using single-cell hydrogel encapsulation and ultrahigh-throughput screening

Engineering catalytic and biophysical properties of enzymes is an essential step en route to advanced biomedical and industrial applications. Here, we developed a high-throughput screening and directed evolution strategy relying on single-cell hydrogel encapsulation to enhance the performance of D -Amino acid oxidase from Rhodotorula gracilis ( Rg DAAOx), a candidate enzyme for cancer therapy. We used a cascade reaction between Rg DAAOx variants surface displayed on yeast and horseradish peroxidase (HRP) in the bulk media to trigger enzyme-mediated crosslinking of phenol-bearing fluorescent alginate macromonomers, resulting in hydrogel formation around single yeast cells. The fluorescent hydrogel capsules served as an artificial phenotype and basis for pooled library screening by fluorescence activated cell sorting (FACS). We screened a Rg DAAOx variant library containing ∼10 6 clones while lowering the D -Ala substrate concentration over three sorting rounds in order to isolate variants with low K m . After three rounds of FACS sorting and regrowth, we isolated and fully characterized four variants displayed on the yeast surface. We identified variants with a more than 5-fold lower K m than the parent sequence, with an apparent increase in substrate binding affinity. The mutations we identified were scattered across the Rg DAAOx structure, demonstrating the difficulty in rationally predicting allosteric sites and highlighting the advantages of scalable library screening technologies for evolving catalytic enzymes

A V-shape superconducting artificial atom based on two inductively coupled transmons

Circuit quantum electrodynamics systems are typically built from resonators and two-level artificial atoms, but the use of multi-level artificial atoms instead can enable promising applications in quantum technology. Here we present an implementation of a Josephson junction circuit dedicated to operate as a V-shape artificial atom. Based on a concept of two internal degrees of freedom, the device consists of two transmon qubits coupled by an inductance. The Josephson nonlinearity introduces a strong diagonal coupling between the two degrees of freedom that finds applications in quantum non-demolition readout schemes, and in the realization of microwave cross-Kerr media based on superconducting circuits.Comment: 5 pages, 3 figure

Optimization of sample-chip design for stub-matched radio-frequency reflectometry measurements

A radio-frequency (rf) matching circuit with an in situ tunable varactor diode used for rf reflectometry measurements in semiconductor nanostructures is investigated and used to optimize the sample-specific chip design. The samples are integrated in a 2-4 GHz stub-matching circuit consisting of a waveguide stub shunted to the terminated coplanar waveguide. Several quantum point contacts fabricated on a GaAs/AlGaAs heterostructure with different chip designs are compared. We show that the change of the reflection coefficient for a fixed change in the quantum point contact conductance can be enhanced by a factor of 3 compared to conventional designs by a suitable electrode geometry

Access to abortion under the heath exception: a comparative analysis in three countries

Background Despite Britain, Colombia, and some Mexican states sharing a health exception within their abortion laws, access to abortion under the health exception varies widely. This study examines factors that result in heterogeneous application of similar health exception laws and consequences for access to legal abortion. Our research adds to previous literature by comparing implementation of similar abortion laws across countries to identify strategies for full implementation of the health exception. Methods We conducted a cross-country comparative descriptive study synthesizing data from document and literature review, official abortion statistics, and interviews with key informants. We gathered information on the use and interpretation of the health exception in the three countries from peer-reviewed literature, court documents, and grey literature. We next extracted public and private abortion statistics to understand the application of the law in each setting. We used a matrix to synthesize information and identify key factors in the use of the law. We conducted in-depth interviews with doctors and experts familiar with the health exception laws in each country and analyzed the qualitative data based on the previously identified factors. Results The health exception is used broadly in Britain, somewhat in Colombia, and very rarely in Mexican states. We identified five factors as particularly salient to application of the health exception in each setting: 1) comprehensiveness of the law including explicit mention of mental health, 2) a strong public health sector that funds abortion, 3) knowledge of and attitudes toward the health exception law, including guidelines for physicians in providing abortion, 4) dissemination of information about the health exception law, and 5) a history of court cases that protect women and clarify the health exception law. Conclusions The health exception is a valuable tool for expanding access to legal abortion. Differences in the use of the health exception as an indication for legal abortion result in wide access for women in Britain to nearly no access in Mexican states. Our findings highlight the difference between theoretical and real access to legal abortion. The interpretation and application of the health exception law are pivotal to expanding real access to abortion

Retinoic Acid-Binding Protein in Human Breast Cancer and Dysplasia

Seventy-five specimens of human breast tissue were checked for the presence of cellular retinoic acid-binding protein (cRABP). Fifty-two percent of the primary carcinomas and 43% of the dysplastic breast lesions (stage MIl) contained detectable amounts of cRABP, whereas no cRABP was found in normal tissue. Sucrose gradient centrifugation and electrophoresis on agarose were used for analysis of the presence of cRABP. The cRABP of human origin (normal uterus and neoplastic mammary tissue) differed in its mobility in agarose electrophoresis from that of rat testis cRA

The most creative organization in the world? The BBC, 'creativity' and managerial style

The managerial styles of two BBC directors-general, John Birt and Greg Dyke, have often been contrasted but not so far analysed from the perspective of their different views of 'creative management'. This article first addresses the orthodox reading of 'Birtism'; second, it locates Dyke's 'creative' turn in the wider context of fashionable neo-management theory and UK government creative industries policy; third, it details Dyke's drive to change the BBC's culture; and finally, it concludes with some reflections on the uncertainties inherent in managing a creative organisation

Expression of phosphate and calcium transporters and their regulators in parotid glands of mice

The concentration of inorganic phosphate (Pi) in plasma is under hormonal control, with deviations from normal values promptly corrected to avoid hyper- or hypophosphatemia. Major regulators include parathyroid hormone (PTH), fibroblast growth factor 23 (FGF-23), and active vitamin D$_{3}$ (calcitriol). This control is achieved by mechanisms largely dependent on regulating intestinal absorption and renal excretion, whose combined actions stabilise plasma Pi levels at around 1–2 mM. Instead, Pi concentrations up to 13 and 40 mM have been measured in saliva from humans and ruminants, respectively, suggesting that salivary glands have the capacity to concentrate Pi. Here we analysed the transcriptome of parotid glands, ileum, and kidneys of mice, to investigate their potential differences regarding the expression of genes responsible for epithelial transport of Pi as well as their known regulators. Given that Pi and Ca$^{2+}$ homeostasis are tightly connected, the expression of genes involved in Ca$^{2+}$ homeostasis was also included. In addition, we studied the effect of vitamin D$_{3}$ treatment on the expression of Pi and Ca$^{2+}$ regulating genes in the three major salivary glands. We found that parotid glands are equipped preferentially with Slc20 rather than with Slc34 Na$^{+}$/Pi cotransporters, are suited to transport Ca$^{2+}$ through the transcellular and paracellular route and are potential targets for PTH and vitamin D$_{3}$ regulation

Functional analysis and transcriptional output of the Göttingen minipig genome

In the past decade the Göttingen minipig has gained increasing recognition as animal model in pharmaceutical and safety research because it recapitulates many aspects of human physiology and metabolism. Genome-based comparison of drug targets together with quantitative tissue expression analysis allows rational prediction of pharmacology and cross-reactivity of human drugs in animal models thereby improving drug attrition which is an important challenge in the process of drug development.; Here we present a new chromosome level based version of the Göttingen minipig genome together with a comparative transcriptional analysis of tissues with pharmaceutical relevance as basis for translational research. We relied on mapping and assembly of WGS (whole-genome-shotgun sequencing) derived reads to the reference genome of the Duroc pig and predict 19,228 human orthologous protein-coding genes. Genome-based prediction of the sequence of human drug targets enables the prediction of drug cross-reactivity based on conservation of binding sites. We further support the finding that the genome of Sus scrofa contains about ten-times less pseudogenized genes compared to other vertebrates. Among the functional human orthologs of these minipig pseudogenes we found HEPN1, a putative tumor suppressor gene. The genomes of Sus scrofa, the Tibetan boar, the African Bushpig, and the Warthog show sequence conservation of all inactivating HEPN1 mutations suggesting disruption before the evolutionary split of these pig species. We identify 133 Sus scrofa specific, conserved long non-coding RNAs (lncRNAs) in the minipig genome and show that these transcripts are highly conserved in the African pigs and the Tibetan boar suggesting functional significance. Using a new minipig specific microarray we show high conservation of gene expression signatures in 13 tissues with biomedical relevance between humans and adult minipigs. We underline this relationship for minipig and human liver where we could demonstrate similar expression levels for most phase I drug-metabolizing enzymes. Higher expression levels and metabolic activities were found for FMO1, AKR/CRs and for phase II drug metabolizing enzymes in minipig as compared to human. The variability of gene expression in equivalent human and minipig tissues is considerably higher in minipig organs, which is important for study design in case a human target belongs to this variable category in the minipig. The first analysis of gene expression in multiple tissues during development from young to adult shows that the majority of transcriptional programs are concluded four weeks after birth. This finding is in line with the advanced state of human postnatal organ development at comparative age categories and further supports the minipig as model for pediatric drug safety studies.; Genome based assessment of sequence conservation combined with gene expression data in several tissues improves the translational value of the minipig for human drug development. The genome and gene expression data presented here are important resources for researchers using the minipig as model for biomedical research or commercial breeding. Potential impact of our data for comparative genomics, translational research, and experimental medicine are discussed