Tukushi modulates Xnr2, FGF, and and BMP signalling: Regulation of Xenopus Germ Layer Formation

BACKGROUND: Cell-cell communication is essential in tissue patterning. In early amphibian development, mesoderm is formed in the blastula-stage embryo through inductive interactions in which vegetal cells act on overlying equatorial cells. Members of the TGF-beta family such as activin B, Vg1, derrière and Xenopus nodal-related proteins (Xnrs) are candidate mesoderm inducing factors, with further activity to induce endoderm of the vegetal region. TGF-beta-like ligands, including BMP, are also responsible for patterning of germ layers. In addition, FGF signaling is essential for mesoderm formation whereas FGF signal inhibition has been implicated in endoderm induction. Clearly, several signaling pathways are coordinated to produce an appropriate developmental output; although intracellular crosstalk is known to integrate multiple pathways, relatively little is known about extracellular coordination. METHODOLOGY/PRINCIPAL FINDINGS: Here, we show that Xenopus Tsukushi (X-TSK), a member of the secreted small leucine rich repeat proteoglycan (SLRP) family, is expressed in ectoderm, endoderm, and the organizer during early development. We have previously reported that X-TSK binds to and inhibits BMP signaling in cooperation with chordin. We now demonstrate two novel interactions: X-TSK binds to and inhibits signaling by FGF8b, in addition to binding to and enhancement of Xnr2 signaling. This signal integration by X-TSK at the extracellular level has an important role in germ layer formation and patterning. Vegetally localized X-TSK potentiates endoderm formation through coordination of BMP, FGF and Xnr2 signaling. In contrast, X-TSK inhibition of FGF-MAPK signaling blocks ventrolateral mesoderm formation, while BMP inhibition enhances organizer formation. These actions of X-TSK are reliant upon its expression in endoderm and dorsal mesoderm, with relative exclusion from ventrolateral mesoderm, in a pattern shaped by FGF signals. CONCLUSIONS/SIGNIFICANCE: Based on our observations, we propose a novel mechanism by which X-TSK refines the field of positional information by integration of multiple pathways in the extracellular space

Patient safety and estimation of renal function in patients prescribed new oral anticoagulants for stroke prevention in atrial fibrillation

OBJECTIVE: In clinical trials of dabigatran and rivaroxaban for stroke prevention in atrial fibrillation (AF), drug eligibility and dosing were determined using the Cockcroft-Gault equation to estimate creatine clearance as a measure of renal function. This cross-sectional study aimed to compare whether using estimated glomerular filtration rate (eGFR) by the widely available and widely used Modified Diet in Renal Disease (MDRD) equation would alter prescribing or dosing of the renally excreted new oral anticoagulants. PARTICIPANTS: Of 4712 patients with known AF within a general practitioner-registered population of 930 079 in east London, data were available enabling renal function to be calculated by both Cockcroft-Gault and MDRD methods in 4120 (87.4%). RESULTS: Of 4120 patients, 2706 were <80 years and 1414 were ≥80 years of age. Among those ≥80 years, 14.9% were ineligible for dabigatran according to Cockcroft-Gault equation but would have been judged eligible applying MDRD method. For those <80 years, 0.8% would have been incorrectly judged eligible for dabigatran and 5.3% would have received too high a dose. For rivaroxaban, 0.3% would have been incorrectly judged eligible for treatment and 13.5% would have received too high a dose. CONCLUSIONS: Were the MDRD-derived eGFR to be used instead of Cockcroft-Gault in prescribing these new agents, many elderly patients with AF would either incorrectly become eligible for them or would receive too high a dose. Safety has not been established using the MDRD equation, a concern since the risk of major bleeding would be increased in patients with unsuspected renal impairment. Given the potentially widespread use of these agents, particularly in primary care, regulatory authorities and drug companies should alert UK doctors of the need to use the Cockcroft-Gault formula to calculate eligibility for and dosing of the new oral anticoagulants in elderly patients with AF and not rely on the MDRD-derived eGFR

Convergence Rate For The Ordered Upwind Method

The ordered upwind method (OUM) is used to approximate the viscosity solution of the static Hamilton---Jacobi---Bellman with direction-dependent weights on unstructured meshes. The method has been previously shown to provide a solution that converges to the exact solution, but no convergence rate has been theoretically proven. In this paper, it is shown that the solutions produced by the OUM in the boundary value formulation converge at a rate of at least the square root of the largest edge length in the mesh in terms of maximum error. An example with similar order of numerical convergence is provided.postprin

Lipid reduction to improve clarification and filterability during primary recovery of intracellular products in yeast lysates using exogenous lipase

BACKGROUND: The yeast Pichia pastoris is a popular host organism for production of a range of biological products, several of which are intracellular. The disruption of yeast cells by homogenisation also releases large quantities of lipids, which can foul the downstream membranes and chromatography matrices used for purification. This work examines lipid removal from yeast cells following homogenisation by enzymatic degradation and its impact on the performance of the subsequent centrifugation and filtration. RESULTS: Lipase treatment of cell homogenate at 37°C for 2 hours, followed by clarification using a scale-down mimic of disc stack centrifugation, resulted in a 6.5-fold improvement in solids removal when compared to untreated feed material. The lipase treated and untreated materials that had undergone initial centrifugation were then tested for filtration performance by passing the material through a 0.45 μm polyethylene sulfone membrane under constant flux. A 50% increase in throughput was observed in comparison to the untreated material. CONCLUSION: This proof-of-concept data suggests enzymatic digestion of lipids, analogous to the widely performed DNA reduction using nucleases, could be a valuable process improvement strategy

Measuring Awareness in People With Dementia: Results of a Systematic Scoping Review

This is the final version. Available on open access from SAGE Publications via the DOI in this recordBackground: Awareness of the diagnosis or related changes in functioning varies in people with dementia (PwD), with implications for the well-being of PwD and their carers. Measuring awareness in a clinical setting could facilitate tailored support and optimize involvement in personal health and care decisions. This scoping review aimed to identify validated methods of assessing awareness in dementia and appraise their clinical utility. Method: A systematic search was conducted of English-language publications that measured awareness in PwD, in 6 electronic databases. Search terms included dement*, Alzheimer*, Pick disease, and awareness, unawareness, anosognosia, insight, denial, metacognit*, or discrepanc*. Results: We screened 30,634 articles, finding 345 articles that met our inclusion criteria. We identified 76 measures, most commonly using a discrepancy questionnaire comparing evaluations of function by PwD and an informant. There were 30 awareness measures developed and validated for use in dementia populations but few designed for general clinical use. Conclusions: Although we found a range of clinical indications for measuring awareness, there were few studies investigating clinical applications and few tools designed for clinical purposes. Further investigation and development of a person-centered tool could facilitate health and care choices in mild-to-moderate dementia.Alzheimer’s Societ

Embryonic/fetal mortality and intrauterine growth restriction is not exclusive to the CBA/J sub-strain in the CBA × DBA model.

Inbred strains of mice are powerful models for understanding human pregnancy complications. For example, the exclusive mating of CBA/J females to DBA/2J males increases fetal resorption to 20-35% with an associated decline in placentation and maintenance of maternal Th1 immunity. More recently other complications of pregnancy, IUGR and preeclampsia, have been reported in this model. The aim of this study was to qualify whether the CBA/CaH substrain female can substitute for CBA/J to evoke a phenotype of embryonic/fetal mortality and IUGR. (CBA/CaH × DBA/2J) F1 had significantly higher embryonic/fetal mortality mortality (p = 0.0063), smaller fetuses (p  10th percentile). In addition, placentae of "normal-weight" (CBA/CaH × DBA/2J) F1 were significantly smaller (p < 0.0006) with a greater proportion of labyrinth (p = 0.0128) and an 11-fold increase in F4/80 + macrophage infiltration (p < 0.0001) when compared to placentae of (CBA/CaH × Balb/c) F1. In conclusion, the embryonic/fetal mortality and IUGR phenotype is not exclusive to CBA/J female mouse, and CBA/CaH females can be substituted to provide a model for the assessment of novel therapeutics

Protocol to analyze and validate transcriptomic changes in PDGFRβ-KO mesenchymal stem cell osteogenic potential in the mouse embryo

Mesenchymal stem/stromal cells (MSCs) can differentiate into osteoblasts under appropriate conditions. PDGFRβ signaling controls MSC osteogenic potential both transcriptomically and in culture. Here, we present a “computer to the bench” protocol to analyze changes in MSC osteogenic potential at transcriptomic and cellular level in the absence of PDGFRβ. We detail the preparation of cells from mouse embryos, the analysis of transcriptomic changes from single-cell RNA-sequencing data, the procedure for MSC derivation and culture, and an osteogenic assay for functional validation. For complete details on the use and execution of this protocol, please refer to Sá da Bandeira et al. (2022).(1

Technical transfer of a rapid microbial platform for vaccine production

The limited availability and affordability of vaccines to low- and middle-income countries (LMIC) has created a need for solutions that will ensure effective, affordable vaccine production technology. To establish a rapid and economical platform for the expression of viral proteins in high yield and purity by Pichia pastoris (X33), the receptor-binding domain (RBD) protein of the SARS-CoV2 was selected in this study. After fermentation at the 5 L scale, the protein was purified by a simplified chromatography, with minimal sample treatment. The purified protein was characterized biochemically, and after its formulation, the immunogenicity was evaluated in mice. Collectively, the data suggested that the vaccine candidate is a suitable COVID-19 vaccine candidate antigen for technology transfer. Furthermore, this study creates a robust foundation for industrial production at scale

Holistic process development to mitigate proteolysis of a subunit rotavirus vaccine candidate produced in Pichia pastoris by means of an acid pH pulse during fed‐batch fermentation

To meet the challenges of global health, vaccine design and development must be reconsidered to achieve cost of goods as low as 15¢ per dose. A new recombinant protein‐based rotavirus vaccine candidate derived from non‐replicative viral subunits fused to a P2 tetanus toxoid CD4(+) T cell epitope is currently under clinical development. We have sought to simplify the existing manufacturing process to meet these aims. To this end, we have taken a holistic process development approach to reduce process complexity and costs while producing a product with the required characteristics. We have changed expression system from Escherichia coli to Pichia pastoris, to produce a secreted product, thereby reducing the number of purification steps. However, the presence of proteases poses challenges to product quality. To understand the effect of fermentation parameters on product quality small‐scale fermentations were carried out. Media pH and fermentation duration had the greatest impact on the proportion of full‐length product. A novel acidic pH pulse strategy was used to minimize proteolysis, and this combined with an early harvest time significantly increased the proportion of full‐length material (60–75%). An improved downstream process using a combination of CIEX and AIEX to further reduce proteases, resulted in maintaining product quality (95% yield)