Sexual dimorphisms in the dermal denticles of thelesser-spotted catshark, Scyliorhinus canicula (Linnaeus, 1758)

The dermal layers of several elasmobranch species have been shown to be sexually dimorphic. Generally, when this occurs the females have thicker dermal layers compared to those of males. This sexual dimorphism has been suggested to occur as a response to male biting during mating. Although male biting as a copulatory behaviour in Scyliorhinus canicula has been widely speculated to occur, only relatively recently has this behaviour been observed. Male S. canicula use their mouths to bite the female's pectoral and caudal fins as part of their pre-copulatory behaviour and to grasp females during copulation. Previous work has shown that female S. canicula have a thicker epidermis compared to that of males. The structure of the dermal denticles in females may also differ from that of males in order to protect against male biting or to provide a greater degree of friction in order to allow the male more purchase. This study reveals that the length, width and density of the dermal denticles of mature male and female S. canicula are sexually dimorphic across the integument in areas where males have been observed to bite and wrap themselves around females (pectoral fin, area posterior to the pectoral fin, caudal fin, and pelvic girdle). No significant differences in the dermal denticle dimensions were found in other body areas examined (head, dorsal skin and caudal peduncle). Sexually dimorphic dermal denticles in mature S. canicula could be a response to male biting/wrapping as part of the copulatory process

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

Molecular quality engineering for low cost vaccine production

Vaccines based on recombinant proteins provide a compelling case for low cost products with broad global accessibility. Protein immunogens are typically derived directly from native sequences found in bacterial or viral pathogens, and may not be well-suited for efficient expression in recombinant hosts. Native immunogens may also suffer from numerous challenges during expression that impact their quality or efficient production, including truncation, aggregation and poor stability. These challenges can lead to inefficiencies in manufacturing of subunit protein vaccines. Typically, recombinant vaccine manufacturing processes are complex, serial batch operations requiring extensive quality testing throughout to ensure product integrity. In response to the Gates Foundation’s Grand Challenge for Innovations in Vaccine Manufacturing for Global Markets, we are co-developing the ULTRA program for flexible, low cost vaccine products. This program aims to develop platform processes for production of recombinant vaccines. We believe that molecular design of the antigens provides a critical handle in improving antigen quality, manufacturability, and product stability, all of which could enable potent, low-cost vaccines. Addressing potential manufacturing challenges early on in product development should enable simple integrated processes for antigen production while minimizing costs associated with quality testing. To this end, we are demonstrating our platform approach with a recombinant trivalent subunit vaccine for rotavirus currently in clinical development. We chose to express the three VP8 subunits in Pichia pastoris to take advantage of the high titers of secreted proteins and minimal process-related contaminants typically experienced with this organism—critical features when developing simple intensified processes to meet our cost targets of $0.15/dose. Initial expression results showed the rotavirus antigens were poorly expressed and suffered from N-terminal truncation and aggregation—all of which were also observed in a previously developed E. coli-based process. We have deployed a two-pronged approach toward improving the manufacturability of these antigens. First, we used a functional genomics approach to identify bottlenecks experienced during cellular expression of the antigens. RNA-sequencing is a mature, inexpensive and acccessible technique for yeast that can indicate host- or sequence-derived bottlenecks in antigen transcription, translation and expression. Second, we made direct sequence changes to the antigens to mitigate specific quality challenges, such as aggregation. Iterations of this approach have enabled robust titers of rotavirus antigens with improved quality. This framework for incorporation of molecular engineering early in development provides a useful model for improving target product profiles that include manufacturability for low-costs, while maintaining immunogenicity

RECTUS FEMORIS MECHANICS IN RUGBY KICKING

This study aimed to quantify rectus femoris muscle-tendon unit length and excitation during different types of rugby kick. Seven male rugby players completed a series of kicks during which kinematic and muscle excitation data were collected. Between 0.2 and 0.1 s prior to ball contact in all kick types, the rectus femoris lengthened rapidly whilst muscle excitation also rapidly increased, identifying eccentric action as a possible mechanism for muscle strain injury. Peak rectus femoris muscle excitations occurred later in the kicks with a primary height demand, and differences in the timing of peak muscle excitation existed between different regions of the rectus femoris muscle. This study provides information which can be used to inform the specificity of physical preparation and rehabilitation protocols for rugby kickers

Identifying the best Pichia pastoris base strain using functional genomics

Market sizes for novel breakthrough therapies and growing demand for existing treatments in emerging markets promise to challenge the current capacity for production of biologics. These trends dictate the need for a concomitant paradigm shift in biomanufacturing toward greater productivity for lower cost. Strain engineering is a promising means to realize the greatest returns by increasing the product titer going into downstream processes. Current cellular hosts are approaching saturation of optimal productivity due to lack of deep biological understanding or limitations of the host’s intrinsic secretion capacity. We demonstrate an approach informed by functional genomics to understand key performance differences between interchangeably-used variants of the host, Pichia pastoris. Genomic variant calling on all USDA-banked and commercially-available strains revealed varying numbers of SNPs relative to the WT strain, Y-11430. Combining transcriptomics and traditional phenotypic assays, the functional impact of these SNPs can inform which host strain is best suited for a given application. Taken together, we have identified key, beneficial SNPs that can be introduced into a WT background to create an IP-free host primed for optimal protein production

Mutation of a conserved, hydrophobic, cryptic epitope improves manufacturability and immunogenicity of the SARS-CoV-2 RBD

The supply of COVID-19 vaccine doses still lags behind the global demand for first time vaccination and booster doses. Distribution of vaccine doses has been far from equitable across the world given the steep prices and logistical challenges that low- and middle-income countries face. Subunit protein vaccine candidates have now been shown to elicit protective responses against SARS-CoV-2 infection, while providing additional benefits for manufacturing capability and stability requirements compared to many currently approved vaccines. Here we report a second-generation engineered RBD sequence variant with enhanced manufacturability and immunogenicity over the wild-type ancestral RBD and a first-generation engineered variant (RBD-L452K-F490W (RBD-J)). Introducing two additional mutations, S383D and L518D, to a hydrophobic cryptic epitope in the RBD core improved expression titers and biophysical stability compared to RBD-J. These two additional mutations in RBD-S383D-L452K-F490W-L518D (RBD-J6) ablated the interaction of two neutralizing antibodies, CR3022 and EY6A, targeting the class 4 epitope on the RBD core, but the protein is still bound by human convalescent sera. Mice immunized with a Beta sequence variant of RBD-J and RBD-J6 displayed on a virus-like particle were protected against challenges with Alpha and Beta variants of SARS-CoV-2. Sera from mice immunized with three doses of a RBD-J6 β – VLP showed comparable neutralizing activity to several variants of concern compared to two doses of Comirnaty. Please click Download on the upper right corner to see the full abstract

The British economy [March 1989]

Evidence is mixed on whether growth in the economy is beginning to turn down. Nevertheless, the growth of GDP is forecast to fall from last year's high of 4.5% to 2.5% this year. Inflationary expectations are the most immediate cause for concern. But following the Budget there is concern that a relatively restrictive fiscal stance may bear down too heavily on output and employment during the next year