Cell Shape Dynamics: From Waves to Migration

We observe and quantify wave-like characteristics of amoeboid migration. Using the amoeba Dictyostelium discoideum, a model system for the study of chemotaxis, we demonstrate that cell shape changes in a wave-like manner. Cells have regions of high boundary curvature that propagate from the leading edge toward the back, usually along alternating sides of the cell. Curvature waves are easily seen in cells that do not adhere to a surface, such as cells that are electrostatically repelled from surfaces or cells that extend over the edge of micro-fabricated cliffs. Without surface contact, curvature waves travel from the leading edge to the back of a cell at ∼35 µm/min. Non-adherent myosin II null cells do not exhibit these curvature waves. At the leading edge of adherent cells, curvature waves are associated with protrusive activity. Like regions of high curvature, protrusive activity travels along the boundary in a wave-like manner. Upon contact with a surface, the protrusions stop moving relative to the surface, and the boundary shape thus reflects the history of protrusive motion. The wave-like character of protrusions provides a plausible mechanism for the zig-zagging of pseudopods and for the ability of cells both to swim in viscous fluids and to navigate complex three dimensional topography

Systems engineering N-glycans of recombinant therapeutic proteins

Protein N-glycosylation reactions form a distributed reaction network spanning over different compartments of Golgi apparatus. The resulting glycan structures are influenced by glycosylation enzymes, the supply rate of nucleotide sugars, as well as competition among extending glycan substrates for a common enzyme and among different enzymes for a common substrate. Controlling the glycan profile of a therapeutic protein product is important for product quality for both innovative drugs as well as biosimilars. Metabolic engineering of the glycosylation pathway offers a venue for modulating the glycan profile. We have taken a systems engineering approach to identify, through model assisted design, the genetic manipulations that may steer the glycan flux to the desired path. However, unlike the energy metabolism pathway for which a small number of enzymes play pivotal roles in controlling the flux, the glycosylation pathway lacks key regulated steps as easily identifiable targets for genetic alteration to re-direct the flux. The model prediction thus serves only as a imprecise guide rather than a clear beacon. Furthermore, very likely multiple genetic alterations are needed in order to steer glycan flux distribution. A scheme of rapid construction of gene combinations to facilitate genetic engineering of the cell is necessary. We establish a golden gate assembly workflow for production of multi-gene constructs for engineering the glycan biosynthesis pathway. Libraries containing promoters of varying strengths, terminators, and glycosylation related coding sequences of interest, all refactored to be devoid of type IIS restriction sites, were synthesized. In the first level of assembly, an additional library of single gene constructs were formed from these base components with single reactions. In the second level of assembly, these monocistrons were then combinatorically combined to form a multi-gene cassette library. In an application of this approach, the N-glycosylation pattern of a recombinant IgG produced in CHO cells was examined with a stoichiometric network visualization tool (GlycoVis) to track the reaction paths which lead to the product glycans and identify galactosylation as potentially limiting glycan maturation. Cassettes consisting of sequences coding for nucleotide sugar synthesis enzymes, nucleotide sugar transporters, and glycosyltransferases were then selected to engineer the IgG producing cell. Multiple cassettes successfully directed the glycosylation to produce antibody with desirable glycoforms. These results served to refine our model parameters and sharpen its predictive capabilities. This combination of systems analysis and synthetic glycoengineering can be broadly applied and enhances our capability to steer N-Glycan patterns and control the quality of therapeutic proteins

Visual communication design: a neglected factor in nutrition promotion via packaged food labels

Packaging design is a communication device and a critical component in branding strategy, and has relevance for food policy. Presently, packaging-related nutrition policy initiatives focus on the role of regulated claims, nutrition information panels and front-of-pack nutrition labels to help guide consumer food choices and address high prevalences of discretionary and ultra-processed food consumption in many countries. However, these nutrition labelling systems are not optimized as public health policy tools as many consumers do not use them to inform their food choices. Visual communication design theory posits that a designer orders the elements and principles of design into hierarchies that prioritize certain elements over others, and that some of these elements are more dominant and given more emphasis than others. The overall design of the package thereby directs consumer attention to some aspects of pack design (e.g., characters, contents of the package) and away from others (e.g., nutrition details). Dual processing frameworks propose that food decisions are made with the interplay between automatic and rational thinking processes. Packaging designs affect whether consumers rely predominantly on automatic or rational thinking to select a food. This narrative review outlines the role of food packaging design and how it impacts the clear communication of nutrition aspects of food products and how the use of nutrition information by consumers to make decisions may depend upon design structures in packaging. This article attests that nutrition scientists and policy makers should incorporate visual communication design into research on the food packaging as a public health promotion tool. A stronger focus on the communication of regulated front-of-pack nutrition information can be made with a re-evaluation of the hierarchy of elements in the front-of-pack design enabling consumers to make healthier decisions

A synthetic biology based cell line engineering pipeline

An ideal host cell line for deriving cell lines of high recombinant protein production should be stable, predictable, and amenable to rapid cell engineering or other forms of phenotypical manipulation. In the past few years we have employed genomic information to identify “safe harbors” for exogenous gene integration in CHO cells, deployed systems modeling and optimization to design pathways and control strategies to modify important aspects of recombinant protein productivity, and established a synthetic biology approach to implement genetic changes, all with the goal of creating a pipeline to produce “designer” cell lines. Chinese hamster ovary (CHO) cells are the preferred platform for protein production. However, the Chinese hamster genome is unstable in its ploidy, is subject to long and short deletions, duplications, and translocations. In addition, gene expression is subject to epigenetic changes including DNA methylation, histone modification and heterochromatin invasion, thus further complicating transgene expression for protein production in cell lines. With these issues in mind, we set out to engineer a CHO cell line highly amenable to stable protein production using a synthetic biology approach. We compiled karyotyping and chromosome number data of several CHO cell lines and sublines, identified genomic regions with high a frequency of gain and loss of copy number using comparative genome hybridization (CGH), and verified structural variants using sequencing data. We further used ATAC (Assay for Transposase-Accessible Chromatin) sequencing to study chromatin accessibility and epigenetic stability within the CHO genome. RNA-seq data from multiple cell lines were also used to identify regions with high transcriptional activity. Analysis of these data allowed the identification of several “safe harbor” loci that could be used for cell engineering. Based on results of the data analysis and identification of “safe harbors”, we engineered an IgG producing cell line with a single copy of the product transgene as a template cell line. This product gene site is flanked by sequences for recombinase mediated cassette exchange, therefore allowing easy substitution of the IgG producing gene for an alternative product gene. Furthermore, a “landing pad” for multi-gene cassette insertion was integrated into the genome at an additional site. Together, these sites allowed engineering of new cell lines producing a fusion protein and Erythropoietin to be generated from the template cell line. To enable rapid assembly of product transgenes and genetic elements for engineering cell attributes into multi-gene cassettes, we adopted a golden-gate based synthetic biology approach. The assembly of genetic parts into multi-gene cassettes in a LEGO-like fashion allowed different combinations of genes under the control of various promoters to be generated quickly for introduction into the template cell line. Using this engineered CHO cell line, we set out to study metabolism and product protein glycosylation for cell engineering. To guide the selection of genetic elements for cell engineering, we developed a multi-compartment kinetic model, as well as a flux model of energy metabolism and glycosylation. The transcriptome meta-data was used extensively to identify genes and isoforms expressed in the cell line and to estimate the enzyme levels in the model. The flux model was used to identify and the LEGO-like platform was used to implement the genetic changes that can alter the glycosylation pattern of the IgG produced by the template cell line. Concurrently we employed a systems optimization approach to identify the genetic alterations in the metabolic pathway to guide cell metabolism toward a favorable state. The model prediction is being implemented experimentally using the synthetic biology approach. In conclusion, we have illustrated a pipeline of rational cell line engineering that integrates genomic science, systems engineering and synthetic biology approaches. The promise, the technical challenges and possible limitations will be discussed in this presentation

CM-Path Molecular Diagnostics Forum-consensus statement on the development and implementation of molecular diagnostic tests in the United Kingdom.

BACKGROUND: Pathology has evolved from a purely morphological description of cellular alterations in disease to our current ability to interrogate tissues with multiple 'omics' technologies. By utilising these techniques and others, 'molecular diagnostics' acts as the cornerstone of precision/personalised medicine by attempting to match the underlying disease mechanisms to the most appropriate targeted therapy. METHODS: Despite the promises of molecular diagnostics, significant barriers have impeded its widespread clinical adoption. Thus, the National Cancer Research Institute (NCRI) Cellular Molecular Pathology (CM-Path) initiative convened a national Molecular Diagnostics Forum to facilitate closer collaboration between clinicians, academia, industry, regulators and other key stakeholders in an attempt to overcome these. RESULTS: We agreed on a consensus 'roadmap' that should be followed during development and implementation of new molecular diagnostic tests. We identified key barriers to efficient implementation and propose possible solutions to these. In addition, we discussed the recent reconfiguration of molecular diagnostic services in NHS England and its likely impacts. CONCLUSIONS: We anticipate that this consensus statement will provide practical advice to those involved in the development of novel molecular diagnostic tests. Although primarily focusing on test adoption within the United Kingdom, we also refer to international guidelines to maximise the applicability of our recommendations

The IDENTIFY study: the investigation and detection of urological neoplasia in patients referred with suspected urinary tract cancer - a multicentre observational study

Objective To evaluate the contemporary prevalence of urinary tract cancer (bladder cancer, upper tract urothelial cancer [UTUC] and renal cancer) in patients referred to secondary care with haematuria, adjusted for established patient risk markers and geographical variation. Patients and Methods This was an international multicentre prospective observational study. We included patients aged ≥16 years, referred to secondary care with suspected urinary tract cancer. Patients with a known or previous urological malignancy were excluded. We estimated the prevalence of bladder cancer, UTUC, renal cancer and prostate cancer; stratified by age, type of haematuria, sex, and smoking. We used a multivariable mixed-effects logistic regression to adjust cancer prevalence for age, type of haematuria, sex, smoking, hospitals, and countries. Results Of the 11 059 patients assessed for eligibility, 10 896 were included from 110 hospitals across 26 countries. The overall adjusted cancer prevalence (n = 2257) was 28.2% (95% confidence interval [CI] 22.3–34.1), bladder cancer (n = 1951) 24.7% (95% CI 19.1–30.2), UTUC (n = 128) 1.14% (95% CI 0.77–1.52), renal cancer (n = 107) 1.05% (95% CI 0.80–1.29), and prostate cancer (n = 124) 1.75% (95% CI 1.32–2.18). The odds ratios for patient risk markers in the model for all cancers were: age 1.04 (95% CI 1.03–1.05; P < 0.001), visible haematuria 3.47 (95% CI 2.90–4.15; P < 0.001), male sex 1.30 (95% CI 1.14–1.50; P < 0.001), and smoking 2.70 (95% CI 2.30–3.18; P < 0.001). Conclusions A better understanding of cancer prevalence across an international population is required to inform clinical guidelines. We are the first to report urinary tract cancer prevalence across an international population in patients referred to secondary care, adjusted for patient risk markers and geographical variation. Bladder cancer was the most prevalent disease. Visible haematuria was the strongest predictor for urinary tract cancer

The role of handedness and statistical learning in adults\u27 language abilities

People use many different strategies in order to learn a language. One way we think individuals learn language is through statistical learning (SL) where they track the regularities in the speech stream in both adjacent (Saffran et al., 1996) and non-adjacent dependencies (NAD) (Gomez, 2002). Handedness is related to adult language abilities (Knecht et al., 2000). This relationship between handedness and language is thought to be driven by lateralization, where handedness is a proxy for lateralization. The current study will investigate the relationship between handedness and SL in adults’ language abilities. Participants will be undergraduate students. Participants\u27 handedness will be assessed with the Waterloo Handedness Questionnaire (Cavill & Bryden, 2003). Their SL will be tested using the SL language from Gomez (2002) testing NADs. Participants will listen to the language during a familiarization period. After listening to the language, participants will hear a string of syllables and will then decide if the string that they heard was correct or incorrect for the language they heard previously. SL abilities will be calculated using the number of syllables they correctly judge as having come from the language they heard previously. It is predicted that lateralized adults, adults who are either left-handed or right-handed, will have better scores on the SL task. A t-test will be used to determine if there was a significant difference in SL scores for left versus right-handed adults. This study will extend and help elucidate some of the mechanisms that may play a role in language

Engaging Boards and Trustees in Strategic Learning: A Toolkit

Effecting social change in a rapidly changing political environment and an increasingly interconnected world requires foundations to adopt a learning orientation. Without continuous learning, grantmakers—and thus boards and trustees—are unaware about what is working where, with whom, and why, as well as what changes or refinements are needed in order to achieve the grantmakers' desired results.This toolkit provides a fresh set of resources for grantmaker CEOs, evaluation staff, and senior leaders to use to engage their boards and trustees in conversations about the importance of strategic learning in their decision-making and deliberation processes