Rational design of expression vectors for high quality biologics

Commercial proteins (e.g. antibodies, enzymes, vaccine components) for applications from biopharmaceuticals to commodity chemicals require low-cost manufacturing of high-quality products. The engineering of recombinant hosts to achieve large quantities of high-quality heterologous proteins is crucial to both minimizing costs and maximizing safety and efficacy (in the case of biopharmaceuticals). High-quality proteins are properly folded and full-length (intact), with native N-, and C-, termini and bear no significant proteolysis or other degradation (oxidation, deamidation, etc…). As most expression hosts rely on recombinant DNA technology for production of the heterologous protein, the transgene cassette provides an early, and inexpensive, opportunity for optimization of quality and protein titer. Commonly, transgene cassettes include a promoter, a heterologous gene of interest, and terminator for expression of the heterologous gene. A targeting sequence for guided recombination and selective marker for isolation of positive clones are also key elements. In engineering the transgene cassette, factors such as the promoter for heterologous gene expression, target site for transgene integration, sequence for translation initiation, and mRNA codon-optimization of the gene of interest are critical design points for a given protein-expressing strain. Here, we demonstrate an approach to transgene cassette optimization in the methylotrophic yeast, Pichia pastoris, informed by functional genomics. Omics-based techniques such as RNA-Seq, ATAC-Seq and ribosomal foot-printing afford greater upfront understanding for subsequent optimized strain engineering on a product-by-product basis. These types of data are cheap and easy to acquire for yeast and can indicate host- or sequence-derived bottlenecks in transgene transcription, translation and expression. Linking these data to product quality attributes can enlighten the design of the expression vector for fast in silico optimization of wide-ranging factors such as gene dosage balance, translation efficiency, and balanced cell kinetics enabling high-quality protein production. Collectively, we show that these tools can enable fast vector design for new heterologous protein-producing strains, including those expressing recombinant vaccines, and robust optimization when engineering higher productivity cell lines

Highly specific PCR-RFLP assays for karyotyping the widespread 2Rb inversion in malaria vectors of the Anopheles gambiae complex

Background: Chromosomal inversion polymorphisms play a role in adaptation to heterogeneous environments. Inversion polymorphisms are implicated in the very high ecological flexibility of the three main malaria vector species of the Afrotropical Anopheles gambiae complex, facilitating the exploitation of anthropogenic environmental modifications and promoting a strong association with humans. In addition to extending the species' spatial and temporal distribution, inversions are associated with epidemiologically relevant mosquito behavior and physiology, underscoring their medical importance. We here present novel PCR-RFLP based assays strongly predictive of genotype for the cosmopolitan 2Rb inversion in An. coluzzii and An. gambiae, a development which overcomes the numerous constraints inherent to traditional cytological karyotyping. Methods: We designed PCR-RFLP genotyping assays based on tag SNPs previously computationally identified as strongly predictive (> 95%) of 2Rb genotype. We targeted those tags whose alternative allelic states destroyed or created the recognition site of a commercially available restriction enzyme, and designed assays with distinctive cleavage profiles for each inversion genotype. The assays were validated on 251 An. coluzzii and 451 An. gambiae cytologically karyotyped specimens from nine countries across Africa and one An. coluzzii laboratory colony. Results: For three tag SNPs, PCR-RFLP assays (denoted DraIII, MspAI, and TatI) reliably produced robust amplicons and clearly distinguishable electrophoretic profiles for all three inversion genotypes. Results obtained with the DraIII assay are ≥ 95% concordant with cytogenetic assignments in both species, while MspAI and TatI assays produce patterns highly concordant with cytogenetic assignments only in An. coluzzii or An. gambiae, respectively. Joint application of species-appropriate pairs of assays increased the concordance levels to > 99% in An. coluzzii and 98% in An. gambiae. Potential sources of discordance (e.g. imperfect association between tag and inversion, allelic dropout, additional polymorphisms in the restriction target site, incomplete or failed restriction digestion) are discussed. Conclusions: The availability of highly specific, cost effective and accessible molecular assays for genotyping 2Rb in An. gambiae and An. coluzzii allows karyotyping of both sexes and all developmental stages. These novel tools will accelerate deeper investigations into the role of this ecologically and epidemiologically important chromosomal inversion in vector biology.[Figure not available: see fulltext.

Parent identity and family-school partnerships: Animating diverse enactments for (special) education decision-making

Family-school partnerships between family members and school personnel can be successful as well as unproductive for parents who have children and youth with developmental disabilities. This qualitative study sought to capture parents’ identities as they negotiated family-school partnerships when making inclusive education decisions and discussing special education service-delivery options for their children and youth with developmental disabilities. Seventeen participants shared their personal narratives in interviews and focus groups. Data were thematically analyzed after an initial round of open-coding generated broad themes. Findings revealed the experiences parents have in partnering with schools span an identity spectrum, including: (a) victim, (b) advocate, (c) perseverer, (d) educator, (e) broker and negotiator, and (f) surrenderer. Implications for policy, practice, and research focus on parent identity and familyschool partnerships

Parents’ experiences in education decision-making for children and youth with disabilities

Families should be an active part of educational decision-making for their children and can be particularly influential in advocating for inclusion for students with intellectual and developmental disabilities. Yet, significant research has shown that parents do not feel schools effectively collaborate with them. We interviewed 19 parents of children with disabilities to investigate the ways they were included and excluded from educational decision-making, and how they decided on their children’s placement and services. Five themes emerged: parents’ exclusion from decision-making, parents’ independent efforts to shape their children’s educational services, parents’ decisions as a result of school and district factors, parents’ role changes to direct their children’s education, and discrepancies between beliefs and experiences of inclusion. Parents’ responses indicate that specific school structures and institutionalized procedures may regularly exclude parents from decision-making. Results have implications for parent-professional partnership during decision-making for students with disabilities and personnel preparation

A Hybrid Sequencing Approach Completes the Genome Sequence of Thermoanaerobacter ethanolicus JW 200

Thermoanaerobacter ethanolicus JW 200 has been identified as a potential sustainable biofuel producer due to its ability to readily ferment carbohydrates to ethanol. A hybrid sequencing approach, combining Oxford Nanopore and Illumina DNA sequence reads, was applied to produce a single contiguous genome sequence of 2,911,280 bp

Improving Outcomes from Breast Cancer in a Low-Income Country: Lessons from Bangladesh

Women in low- and middle-income countries (LMICs) have yet to benefit from recent advances in breast cancer diagnosis and treatment now experienced in high-income countries. Their unique sociocultural and health system circumstances warrant a different approach to breast cancer management than that applied to women in high-income countries. Here, we present experience from the last five years working in rural Bangladesh. Case and consecutive series data, focus group and individual interviews, and clinical care experience provide the basis for this paper. These data illustrate a complex web of sociocultural, economic, and health system conditions which affect womens' choices to seek and accept care and successful treatment. We conclude that health system, human rights, and governance issues underlie high mortality from this relatively rare disease in Bangladesh

Twirling Elastica: Kinks, Viscous Drag, and Torsional Stress

Biological filaments such as DNA or bacterial flagella are typically curved in their natural states. To elucidate the interplay of viscous drag, twisting, and bending in the overdamped dynamics of such filaments, we compute the steady-state torsional stress and shape of a rotating rod with a kink. Drag deforms the rod, ultimately extending or folding it depending on the kink angle. For certain kink angles and kink locations, both states are possible at high rotation rates. The agreement between our macroscopic experiments and the theory is good, with no adjustable parameters.Comment: 4 pages, 4 figure

Developing a quality assurance metric: a panoptic view

This article is a post-print of the published article that may be accessed at the link below. Copyright @ 2006 Sage Publications.There are a variety of techniques that lecturers can use to get feedback on their teaching - for example, module feedback and coursework results. However, a question arises about how reliable and valid are the content that goes into these quality assurance metrics. The aim of this article is to present a new approach for collecting and analysing qualitative feedback from students that could be used as the first stage in developing more reliable quality assurance metrics. The approach, known as the multi-dimensional crystal view, is based on the belief that individuals have different views on the benefits that the embedded process in a system can have on the behaviour of the system. The results of this study indicate that in the context of evaluation and feedback methods, the multi-dimensional approach appears to provide the opportunity for developing more effective student feedback mechanisms

Stretching Instability of Helical Spring

We show that when a gradually increasing tensile force is applied to the ends of a helical spring with sufficiently large ratios of radius to pitch and twist to bending rigidity, the end-to-end distance undergoes a sequence of discontinuous stretching transitions. Subsequent decrease of the force leads to step-like contraction and hysteresis is observed. For finite helices, the number of these transitions increases with the number of helical turns but only one stretching and one contraction instability survive in the limit of an infinite helix. We calculate the critical line that separates the region of parameters in which the deformation is continuous from that in which stretching instabilities occur, and propose experimental tests of our predictions.Comment: 5 pages, 4 figure