A Fast Alternative to Soft Lithography for the Fabrication of Organ-on-a-Chip Elastomeric-Based Devices and Microactuators

Organ-on-a-chip technology promises to revolutionize how pre-clinical human trials are conducted. Engineering an in vitro environment that mimics the functionality and architecture of human physiology is essential toward building better platforms for drug development and personalized medicine. However, the complex nature of these devices requires specialized, time consuming, and expensive fabrication methodologies. Alternatives that reduce design-to-prototype time are needed, in order to fulfill the potential of these devices. Here, a streamlined approach is proposed for the fabrication of organ-on-a-chip devices with incorporated microactuators, by using an adaptation of xurography. This method can generate multilayered, membrane-integrated biochips in a matter of hours, using low-cost benchtop equipment. These devices are capable of withstanding considerable pressure without delamination. Furthermore, this method is suitable for the integration of flexible membranes, required for organ-on-a-chip applications, such as mechanical actuation or the establishment of biological barrier function. The devices are compatible with cell culture applications and present no cytotoxic effects or observable alterations on cellular homeostasis. This fabrication method can rapidly generate organ-on-a-chip prototypes for a fraction of cost and time, in comparison to conventional soft lithography, constituting an interesting alternative to the current fabrication methods.C.O. and P.L.G. contributed equally to this work as co‐senior authors. This work was supported by Fundação para a Ciência e Tecnologia (FCT) and Doctoral Programme on Cellular and Molecular Biotechnology Applied to Health Sciences (BiotechHealth Programme; ref. PD/00016/2012), by Programa Operacional Potencial Humano (POPH), and SkinChip project (PTDC/BBB‐BIO/1889/2014). The work has been also financed by: 1) Fundo Europeu de Desenvolvimento (FEDER) Regional funds through the COMPETE 2020 – Operacional Programme for Competitiveness and Internationalization (POCI), Portugal 2020, and by Portuguese funds through FCT/Ministério da Ciência, Tecnologia e Inovação in the framework of the projects “Institute for Research and Innovation in Health Sciences” (POCI‐01‐0145‐FEDER‐007274), 3DChroMe (PTDC/BTM‐TEC/30164/2017); Norte Portugal Regional Programme (NORTE 2020), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (ERDF) for projects NORTE‐01‐0145‐FEDER‐000029 and DOCnet (NORTE‐01‐0145‐FEDER‐000003). D.A.F. acknowledges FCT for his support through a FCT/BiotechHealth PhD Programme grant, ref. PD/BD/105976/2014. J.P.C. acknowledges funding from the European Structural and Investment funds through the Compete Programme (Grant #: LISBOA‐01‐0145‐FEDER‐016405) and from National funds through FCT (SAICTPAC/0019/2015) via the research project POINT4PAC, and FCT funding through INESC MN (Unidade ID 5367). The authors would also like to thank: Jorge Ferreira (Chromosome Instability Group, i3S/IBMC) for granting access to the plasma cleaner equipment and for the insightful scientific support; i3S Scientific Platform (Biointerfaces and Nanotechnology core facility, i3S/INEB), member of the national infrastructure PPBI – Portuguese Platform of Bioimaging (PPBI‐POCI‐01‐0145‐FEDER‐022122), in particular Maria Lázaro for support and access to the SP5 confocal microscope; Aureliana Sousa (Biofabrication Group at i3S/INEB) for scientific support and discussion; Dina Leitão (Centro Hospitalar e Universitário São João) for providing access to the normal gastric mucosa specimens; Celso Reis for kindly providing the antibody against Mucin‐1. C.O. and P.L.G. contributed equally to this work as co-senior authors. This work was supported by Funda??o para a Ci?ncia e Tecnologia (FCT) and Doctoral Programme on Cellular and Molecular Biotechnology Applied to?Health Sciences (BiotechHealth Programme; ref.?PD/00016/2012),?by Programa Operacional Potencial Humano (POPH), and SkinChip project (PTDC/BBB-BIO/1889/2014). The work has been also financed by: 1) Fundo Europeu de Desenvolvimento (FEDER) Regional funds through the COMPETE 2020 ? Operacional Programme for Competitiveness and Internationalization (POCI), Portugal 2020, and by Portuguese funds through FCT/Minist?rio da Ci?ncia, Tecnologia e Inova??o in the framework of the projects ?Institute for Research and Innovation in Health Sciences? (POCI-01-0145-FEDER-007274), 3DChroMe (PTDC/BTM-TEC/30164/2017); Norte Portugal Regional Programme (NORTE 2020), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (ERDF) for projects NORTE-01-0145-FEDER-000029 and DOCnet (NORTE-01-0145-FEDER-000003). D.A.F. acknowledges FCT for his support through a FCT/BiotechHealth PhD Programme grant, ref. PD/BD/105976/2014. J.P.C. acknowledges funding from the European Structural and Investment funds through the Compete Programme (Grant #: LISBOA-01-0145-FEDER-016405) and from National funds through FCT (SAICTPAC/0019/2015) via the research project POINT4PAC, and FCT funding through INESC MN (Unidade ID 5367). The authors would also like to thank: Jorge Ferreira (Chromosome Instability Group, i3S/IBMC) for granting access to the plasma cleaner equipment and for the insightful scientific support; i3S Scientific Platform (Biointerfaces and Nanotechnology core facility, i3S/INEB), member of the national infrastructure PPBI ? Portuguese Platform of Bioimaging (PPBI-POCI-01-0145-FEDER-022122), in particular Maria L?zaro for support and access to the SP5 confocal microscope; Aureliana Sousa (Biofabrication Group at i3S/INEB) for scientific support and discussion; Dina Leit?o (Centro Hospitalar e Universit?rio S?o Jo?o) for providing access to the normal gastric mucosa specimens; Celso Reis for kindly providing the antibody against Mucin-1

Phosphorylation-dependent assembly and coordination of the DNA damage checkpoint apparatus by Rad4TopBP1

The BRCT-domain protein Rad4(TopBP1) facilitates activation of the DNA damage checkpoint in Schizosaccharomyces pombe by physically coupling the Rad9-Rad1-Hus1 clamp, the Rad3(ATR) -Rad26(ATRIP) kinase complex, and the Crb2(53BP1) mediator. We have now determined crystal structures of the BRCT repeats of Rad4(TopBP1), revealing a distinctive domain architecture, and characterized their phosphorylation-dependent interactions with Rad9 and Crb2(53BP1). We identify a cluster of phosphorylation sites in the N-terminal region of Crb2(53BP1) that mediate interaction with Rad4(TopBP1) and reveal a hierarchical phosphorylation mechanism in which phosphorylation of Crb2(53BP1) residues Thr215 and Thr235 promotes phosphorylation of the noncanonical Thr187 site by scaffolding cyclin-dependent kinase (CDK) recruitment. Finally, we show that the simultaneous interaction of a single Rad4(TopBP1) molecule with both Thr187 phosphorylation sites in a Crb2(53BP1) dimer is essential for establishing the DNA damage checkpoint

Crystal structure of the Ego1-Ego2-Ego3 complex and its role in promoting Rag GTPase-dependent TORC1 signaling

The target of rapamycin complex 1 (TORC1) integrates various hormonal and nutrient signals to regulate cell growth, proliferation, and differentiation. Amino acid-dependent activation of TORC1 is mediated via the yeast EGO complex (EGOC) consisting of Gtr1, Gtr2, Ego1, and Ego3. Here, we identify the previously uncharacterized Ycr075w-a/Ego2 protein as an additional EGOC component that is required for the integrity and localization of the heterodimeric Gtr1-Gtr2 GTPases, equivalent to mammalian Rag GTPases. We also report the crystal structure of the Ego1-Ego2-Ego3 ternary complex (EGO-TC) at 2.4 Å resolution, in which Ego2 and Ego3 form a heterodimer flanked along one side by Ego1. Structural data also reveal the structural conservation of protein components between the yeast EGO-TC and the human Ragulator, which acts as a GEF for Rag GTPases. Interestingly, however, artificial tethering of Gtr1-Gtr2 to the vacuolar membrane is sufficient to activate TORC1 in response to amino acids even in the absence of the EGO-TC. Our structural and functional data therefore support a model in which the EGO-TC acts as a scaffold for Rag GTPases in TORC1 signaling

Varieties of crisis and working conditions: A comparative study between Greece and Serbia

We explore two historically different, yet regionally connected, countries and the way that their weak institutional foundations and long-term economic turbulence have made them unable to overcome crises, leading to the institutionalisation of adverse working conditions. We focus on the outcomes of the systemic crisis in Greece and the transition crisis in Serbia using semi-structured interviews and focus groups with managers and employees in small and medium-sized enterprises (SMEs) in two time periods. We argue that, although the crisis has different origins in the two countries, in both it has led to adverse working conditions becoming institutionalised in organisations and, therefore, less likely to change. Our research explores the institutionalisation of adverse working conditions and offers an understanding of the lived reality of institutions in the way they are experienced by individuals, examining variations in the origins, pressures and outcomes of different types of crises on business practices from an individual perspective

Direct Injection of Functional Single-Domain Antibodies from E. coli into Human Cells

Intracellular proteins have a great potential as targets for therapeutic antibodies (Abs) but the plasma membrane prevents access to these antigens. Ab fragments and IgGs are selected and engineered in E. coli and this microorganism may be also an ideal vector for their intracellular delivery. In this work we demonstrate that single-domain Ab (sdAbs) can be engineered to be injected into human cells by E. coli bacteria carrying molecular syringes assembled by a type III protein secretion system (T3SS). The injected sdAbs accumulate in the cytoplasm of HeLa cells at levels ca. 105–106 molecules per cell and their functionality is shown by the isolation of sdAb-antigen complexes. Injection of sdAbs does not require bacterial invasion or the transfer of genetic material. These results are proof-of-principle for the capacity of E. coli bacteria to directly deliver intracellular sdAbs (intrabodies) into human cells for analytical and therapeutic purposes

Strategic and practical guidelines for successful structured illumination microscopy

Linear 2D- or 3D-structured illumination microscopy (SIM or3D-SIM, respectively) enables multicolor volumetric imaging of fixed and live specimens with subdiffraction resolution in all spatial dimensions. However, the reliance of SIM on algorithmic post-processing renders it particularly sensitive to artifacts that may reduce resolution, compromise data and its interpretations, and drain resources in terms of money and time spent. Here we present a protocol that allows users to generate high-quality SIM data while accounting and correcting for common artifacts. The protocol details preparation of calibration bead slides designed for SIM-based experiments, the acquisition of calibration data, the documentation of typically encountered SIM artifacts and corrective measures that should be taken to reduce them. It also includes a conceptual overview and checklist for experimental design and calibration decisions, and is applicable to any commercially available or custom platform. This protocol, plus accompanying guidelines, allows researchers from students to imaging professionals to create an optimal SIM imaging environment regardless of specimen type or structure of interest. The calibration sample preparation and system calibration protocol can be executed within 1-2 d