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

“That Looks Like Me or Something i Can Do”: Affordances and Constraints in the Online Identity Work of US LGBTQ+ Millennials

This article examines how search engines and social networking sites enable and constrain the identity-related information practices of lesbian, gay, bisexual, transgender,and queer (LGBTQ+) millennials in the United States

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

CpG-Methylation Regulates a Class of Epstein-Barr Virus Promoters

DNA methylation is the major modification of eukaryotic genomes and plays an essential role in mammalian gene regulation. In general, cytosine-phosphatidyl-guanosine (CpG)-methylated promoters are transcriptionally repressed and nuclear proteins such as MECP2, MBD1, MBD2, and MBD4 bind CpG-methylated DNA and contribute to epigenetic silencing. Methylation of viral DNA also regulates gene expression of Epstein-Barr virus (EBV), which is a model of herpes virus latency. In latently infected human B cells, the viral DNA is CpG-methylated, the majority of viral genes is repressed and virus synthesis is therefore abrogated. EBV's BZLF1 encodes a transcription factor of the AP-1 family (Zta) and is the master gene to overcome viral gene repression. In a genome-wide screen, we now identify and characterize those viral genes, which Zta regulates. Among them are genes essential for EBV's lytic phase, which paradoxically depend on strictly CpG-methylated promoters for their Zta-induced expression. We identified novel DNA recognition motifs, termed meZRE (methyl-Zta-responsive element), which Zta selectively binds in order to ‘read’ DNA in a methylation- and sequence-dependent manner unlike any other known protein. Zta is a homodimer but its binding characteristics to meZREs suggest a sequential, non-palindromic and bipartite DNA recognition element, which confers superior DNA binding compared to CpG-free ZREs. Our findings indicate that Zta has evolved to transactivate cytosine-methylated, hence repressed, silent promoters as a rule to overcome epigenetic silencing

FHL2 interacts with CALM and is highly expressed in acute erythroid leukemia

The t(10;11)(p13;q14) translocation results in the fusion of the CALM (clathrin assembly lymphoid myeloid leukemia protein) and AF10 genes. This translocation is observed in acute myeloblastic leukemia (AML M6), acute lymphoblastic leukemia (ALL) and malignant lymphoma. Using a yeast two-hybrid screen, the four and a half LIM domain protein 2 (FHL2) was identified as a CALM interacting protein. Recently, high expression of FHL2 in breast, gastric, colon, lung as well as in prostate cancer was shown to be associated with an adverse prognosis. The interaction between CALM and FHL2 was confirmed by glutathione S-transferase-pulldown assay and co-immunoprecipitation experiments. The FHL2 interaction domain of CALM was mapped to amino acids 294–335 of CALM. The transcriptional activation capacity of FHL2 was reduced by CALM, but not by CALM/AF10, which suggests that regulation of FHL2 by CALM might be disturbed in CALM/AF10-positive leukemia. Extremely high expression of FHL2 was seen in acute erythroid leukemia (AML M6). FHL2 was also highly expressed in chronic myeloid leukemia and in AML with complex aberrant karyotype. These results suggest that FHL2 may play an important role in leukemogenesis, especially in the case of AML M6

Recruitment of the mitotic exit network to yeast centrosomes couples septin displacement to actomyosin constriction

The Mitotic Exit Network (MEN) promotes mitotic exit and cytokinesis but if and how MEN independently controls these two processes is unclear. Here, the authors report that MEN displaces septins from the cell division site to promote actomyosin ring constriction, independently of MEN control of mitotic exit

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

Single domain antibodies: promising experimental and therapeutic tools in infection and immunity

Antibodies are important tools for experimental research and medical applications. Most antibodies are composed of two heavy and two light chains. Both chains contribute to the antigen-binding site which is usually flat or concave. In addition to these conventional antibodies, llamas, other camelids, and sharks also produce antibodies composed only of heavy chains. The antigen-binding site of these unusual heavy chain antibodies (hcAbs) is formed only by a single domain, designated VHH in camelid hcAbs and VNAR in shark hcAbs. VHH and VNAR are easily produced as recombinant proteins, designated single domain antibodies (sdAbs) or nanobodies. The CDR3 region of these sdAbs possesses the extraordinary capacity to form long fingerlike extensions that can extend into cavities on antigens, e.g., the active site crevice of enzymes. Other advantageous features of nanobodies include their small size, high solubility, thermal stability, refolding capacity, and good tissue penetration in vivo. Here we review the results of several recent proof-of-principle studies that open the exciting perspective of using sdAbs for modulating immune functions and for targeting toxins and microbes

Conformational Targeting of Fibrillar Polyglutamine Proteins in Live Cells Escalates Aggregation and Cytotoxicity

Misfolding- and aggregation-prone proteins underlying Parkinson's, Huntington's and Machado-Joseph diseases, namely alpha-synuclein, huntingtin, and ataxin-3 respectively, adopt numerous intracellular conformations during pathogenesis, including globular intermediates and insoluble amyloid-like fibrils. Such conformational diversity has complicated research into amyloid-associated intracellular dysfunction and neurodegeneration. To this end, recombinant single-chain Fv antibodies (scFvs) are compelling molecular tools that can be selected against specific protein conformations, and expressed inside cells as intrabodies, for investigative and therapeutic purposes.Using atomic force microscopy (AFM) and live-cell fluorescence microscopy, we report that a human scFv selected against the fibrillar form of alpha-synuclein targets isomorphic conformations of misfolded polyglutamine proteins. When expressed in the cytoplasm of striatal cells, this conformation-specific intrabody co-localizes with intracellular aggregates of misfolded ataxin-3 and a pathological fragment of huntingtin, and enhances the aggregation propensity of both disease-linked polyglutamine proteins. Using this intrabody as a tool for modulating the kinetics of amyloidogenesis, we show that escalating aggregate formation of a pathologic huntingtin fragment is not cytoprotective in striatal cells, but rather heightens oxidative stress and cell death as detected by flow cytometry. Instead, cellular protection is achieved by suppressing aggregation using a previously described intrabody that binds to the amyloidogenic N-terminus of huntingtin. Analogous cytotoxic results are observed following conformational targeting of normal or polyglutamine-expanded human ataxin-3, which partially aggregate through non-polyglutamine domains.These findings validate that the rate of aggregation modulates polyglutamine-mediated intracellular dysfunction, and caution that molecules designed to specifically hasten aggregation may be detrimental as therapies for polyglutamine disorders. Moreover, our findings introduce a novel antibody-based tool that, as a consequence of its general specificity for fibrillar conformations and its ability to function intracellularly, offers broad research potential for a variety of human amyloid diseases