Precise, high-throughput production of multicellular spheroids with a bespoke 3D bioprinter

3D in vitro cancer models are important therapeutic and biological discovery tools, yet formation of multicellular spheroids in a throughput and highly controlled manner to achieve robust and statistically relevant data, remains challenging. Here, we developed an enabling technology consisting of a bespoke drop-on-demand 3D bioprinter capable of high-throughput printing of 96-well plates of spheroids. 3D-multicellular spheroids are embedded inside a tissue-like matrix with precise control over size and cell number. Application of 3D bioprinting for high-throughput drug screening was demonstrated with doxorubicin. Measurements showed that IC 50 values were sensitive to spheroid size, embedding and how spheroids conform to the embedding, revealing parameters shaping biological responses in these models. Our study demonstrates the potential of 3D bioprinting as a robust high-throughput platform to screen biological and therapeutic parameters. Significance Statement In vitro 3D cell cultures serve as more realistic models, compared to 2D cell culture, for understanding diverse biology and for drug discovery. Preparing 3D cell cultures with defined parameters is challenging, with significant failure rates when embedding 3D multicellular spheroids into extracellular mimics. Here, we report a new 3D bioprinter we developed in conjunction with bioinks to allow 3D-multicellular spheroids to be produced in a high-throughput manner. High-throughput production of embedded multicellular spheroids allowed entire drug-dose responses to be performed in 96-well plate format with statistically relevant numbers of data points. We have deconvoluted important parameters in drug responses including the impact of spheroid size and embedding in an extracellular matrix mimic on IC 50 values

Diagnostic Impact of Repeated Expert Review & Long‐Term Follow‐Up in Determining Etiology of Idiopathic Cardiac Arrest

Background: Recognizing the etiology of sudden cardiac arrest (SCA) has an enormous impact on the management of victims and their immediate families. A significant proportion of SCA survivors with a structurally normal heart are not offered a diagnosis and there is no clear consensus on the type and duration of follow‐up. We aimed to assess the utility of a multidisciplinary approach in optimizing diagnosis of cardiac arrest etiology during follow‐up. Methods and Results: We retrospectively assessed 327 consecutive SCA survivors (mean age 61.9±16.2 years, 80% men) who underwent secondary prevention implantable cardioverter defibrillators between May 2015 and November 2018. The initial diagnosis was recorded at the time of admission and follow‐up diagnosis was deduced from subsequent clinic records, investigations, and outcomes of multidisciplinary team meetings. Structural heart disease accounted for 282 (86%) of SCAs. Forty‐five (14%) patients had a structurally normal heart and underwent comprehensive testing and follow‐up (mean duration 93±52 weeks). On initial evaluation, 14/45 (31%) of these received a diagnosis, rising to 29/45 (64%) with serial reviews during follow‐up. Discussion in multidisciplinary team meetings and imaging reassessment accounted for 47% of new diagnoses. No additional diagnoses were made beyond 96 weeks. Nineteen (5.8%) fatalities occurred in the entire cohort, exclusively in patients with structural heart disease. Conclusions: Systematic comprehensive testing combined with multidisciplinary expert team review of SCA survivors without structural heart disease improves the yield and time to diagnosis compared with previously published studies. This approach has positive implications in the management of SCA survivors and their families

Low noise amplication of an optically carried microwave signal: application to atom interferometry

In this paper, we report a new scheme to amplify a microwave signal carried on a laser light at $\lambda$=852nm. The amplification is done via a semiconductor tapered amplifier and this scheme is used to drive stimulated Raman transitions in an atom interferometer. Sideband generation in the amplifier, due to self-phase and amplitude modulation, is investigated and characterized. We also demonstrate that the amplifier does not induce any significant phase-noise on the beating signal. Finally, the degradation of the performances of the interferometer due to the amplification process is shown to be negligible

Industrial constructions of publics and public knowledge: a qualitative investigation of practice in the UK chemicals industry

This is a post print version of the article. The official published version can be obtained from the link below - © 2007 by SAGE PublicationsWhile the rhetoric of public engagement is increasingly commonplace within industry, there has been little research that examines how lay knowledge is conceptualized and whether it is really used within companies. Using the chemicals sector as an example, this paper explores how companies conceive of publics and "public knowledge," and how this relates to modes of engagement/communication with them. Drawing on qualitative empirical research in four companies, we demonstrate that the public for industry are primarily conceived as "consumers" and "neighbours," having concerns that should be allayed rather than as groups with knowledge meriting engagement. We conclude by highlighting the dissonance between current advocacy of engagement and the discourses and practices prevalent within industry, and highlight the need for more realistic strategies for industry/public engagement.Funding was received from the ESRC Science in Society Programme

Is This the Prime Time for Transradial Access Left Ventricular Endomyocardial Biopsy?

Left ventricular endomyocardial biopsy (EMB) is an essential tool in the management of myocarditis and is conventionally performed via transfemoral access (TFA). Transradial access EMB (TRA-EMB) is a novel alternative and the authors sought to determine its safety and feasibility by conducting a systematic review of the literature. Medline was searched in 2020, and cohort demographics, procedural details and complications were extracted from selected studies. Four observational studies with a combined total of 496 procedures were included. TRA-EMB was most frequently performed with a sheathless MP1 guide catheter via the right radial artery. The most common complication was pericardial effusion (up to 11% in one study), but pericardial drainage for tamponade was rare (one reported case). Death and mitral valve damage have not been reported. TRA-EMB was successful in obtaining samples in 99% of reported procedures. The authors concluded that TRA-EMB is a safe and feasible alternative to TFA-EMB and the most common complication is uncomplicated pericardial effusion

A covalently crosslinked bioink for multi-materials drop-on-demand 3D bioprinting of three-dimensional cell cultures

In vitro three-dimensional (3D) cell models have been accepted to better recapitulate aspects of in vivo organ environment than 2D cell culture. Currently, the production of these complex in vitro 3D cell models with multiple cell types and microenvironments remains challenging and prone to human error. Here we report a versatile bioink comprised of a 4-arm PEG based polymer with distal maleimide derivatives as the main ink component and a bis-thiol species as the activator that crosslinks the polymer to form the hydrogel in less than a second. The rapid gelation makes the polymer system compatible with 3D bioprinting. The ink is combined with a drop-on-demand 3D bioprinting platform consisting of eight independently addressable nozzles and high-throughput printing logic for creating complex 3D cell culture models. The combination of multiple nozzles and fast printing logic enables the rapid preparation of many complex 3D structures comprising multiple hydrogel environments in the one structure in a standard 96-well plate format. The platform compatibility for biological applications was validated using pancreatic ductal adenocarcinoma cancer (PDAC) cells with their phenotypic responses controlled by tuning the hydrogel microenvironment

Do advanced glycation end products contribute to food allergy?

Sugars can bind non-enzymatically to proteins, nucleic acids or lipids and form compounds called Advanced Glycation End Products (AGEs). Although AGEs can form in vivo, factors in the Western diet such as high amounts of added sugars, processing methods such as dehydration of proteins, high temperature sterilisation to extend shelf life, and cooking methods such as frying and microwaving (and reheating), can lead to inordinate levels of dietary AGEs. Dietary AGEs (dAGEs) have the capacity to bind to the Receptor for Advanced Glycation End Products (RAGE) which is part of the endogenous threat detection network. There are persuasive epidemiological and biochemical arguments that correlate the rise in food allergy in several Western countries with increases in dAGEs. The increased consumption of dAGEs is enmeshed in current theories of the aetiology of food allergy which will be discussed

Intermanifold similarities in partial photoionization cross sections of helium

Using the eigenchannel R-matrix method we calculate partial photoionization cross sections from the ground state of the helium atom for incident photon energies up to the N=9 manifold. The wide energy range covered by our calculations permits a thorough investigation of general patterns in the cross sections which were first discussed by Menzel and co-workers [Phys. Rev. A {\bf 54}, 2080 (1996)]. The existence of these patterns can easily be understood in terms of propensity rules for autoionization. As the photon energy is increased the regular patterns are locally interrupted by perturber states until they fade out indicating the progressive break-down of the propensity rules and the underlying approximate quantum numbers. We demonstrate that the destructive influence of isolated perturbers can be compensated with an energy-dependent quantum defect.Comment: 10 pages, 10 figures, replacement with some typos correcte

Mass spectrometry imaging identifies palmitoylcarnitine as an immunological mediator during Salmonella Typhimurium infection

Salmonella Typhimurium causes a self-limiting gastroenteritis that may lead to systemic disease. Bacteria invade the small intestine, crossing the intestinal epithelium from where they are transported to the mesenteric lymph nodes (MLNs) within migrating immune cells. MLNs are an important site at which the innate and adaptive immune responses converge but their architecture and function is severely disrupted during S. Typhimurium infection. To further understand host-pathogen interactions at this site, we used mass spectrometry imaging (MSI) to analyse MLN tissue from a murine model of S. Typhimurium infection. A molecule, identified as palmitoylcarnitine (PalC), was of particular interest due to its high abundance at loci of S. Typhimurium infection and MLN disruption. High levels of PalC localised to sites within the MLNs where B and T cells were absent and where the perimeter of CD169+ sub capsular sinus macrophages was disrupted. MLN cells cultured ex vivo and treated with PalC had reduced CD4+CD25+ T cells and an increased number of B220+CD19+ B cells. The reduction in CD4+CD25+ T cells was likely due to apoptosis driven by increased caspase-3/7 activity. These data indicate that PalC significantly alters the host response in the MLNs, acting as a decisive factor in infection outcome

The Efficacy of Sunitinib Treatment of Renal Cancer Cells Is Associated with the Protein PHAX In Vitro

Anti-angiogenic agents, such as the multi-tyrosine kinase inhibitor sunitinib, are key first line therapies for metastatic clear cell renal cell carcinoma (ccRCC), but their mechanism of action is not fully understood. Here, we take steps towards validating a computational prediction based on differential transcriptome network analysis that phosphorylated adapter RNA export protein (PHAX) is associated with sunitinib drug treatment. The regulatory impact factor differential network algorithm run on patient tissue samples suggests PHAX is likely an important regulator through changes in genome-wide network connectivity. Immunofluorescence staining of patient tumours showed strong localisation of PHAX to the microvasculature consistent with the anti-angiogenic effect of sunitinib. In normal kidney tissue, PHAX protein abundance was low but increased with tumour grade (G1 vs. G3/4; p 0.01), consistent with a possible role in cancer progression. In organ culture, ccRCC cells had higher levels of PHAX protein expression than normal kidney cells, and sunitinib increased PHAX protein expression in a dose dependent manner (untreated vs. 100 µM; p 0.05). PHAX knockdown in a ccRCC organ culture model impacted the ability of sunitinib to cause cancer cell death (p 0.0001 untreated vs. treated), suggesting a role for PHAX in mediating the efficacy of sunitinib