Direct and dynamic detection of HIV-1 in living cells.

In basic and applied HIV research, reliable detection of viral components is crucial to monitor progression of infection. While it is routine to detect structural viral proteins in vitro for diagnostic purposes, it previously remained impossible to directly and dynamically visualize HIV in living cells without genetic modification of the virus. Here, we describe a novel fluorescent biosensor to dynamically trace HIV-1 morphogenesis in living cells. We generated a camelid single domain antibody that specifically binds the HIV-1 capsid protein (CA) at subnanomolar affinity and fused it to fluorescent proteins. The resulting fluorescent chromobody specifically recognizes the CA-harbouring HIV-1 Gag precursor protein in living cells and is applicable in various advanced light microscopy systems. Confocal live cell microscopy and super-resolution microscopy allowed detection and dynamic tracing of individual virion assemblies at the plasma membrane. The analysis of subcellular binding kinetics showed cytoplasmic antigen recognition and incorporation into virion assembly sites. Finally, we demonstrate the use of this new reporter in automated image analysis, providing a robust tool for cell-based HIV research

Differential recruitment of DNA Ligase I and III to DNA repair sites

DNA ligation is an essential step in DNA replication, repair and recombination. Mammalian cells contain three DNA Ligases that are not interchangeable although they use the same catalytic reaction mechanism. To compare the recruitment of the three eukaryotic DNA Ligases to repair sites in vivo we introduced DNA lesions in human cells by laser microirradiation. Time lapse microscopy of fluorescently tagged proteins showed that DNA Ligase III accumulated at microirradiated sites before DNA Ligase I, whereas we could detect only a faint accumulation of DNA Ligase IV. Recruitment of DNA Ligase I and III to repair sites was cell cycle independent. Mutational analysis and binding studies revealed that DNA Ligase I was recruited to DNA repair sites by interaction with PCNA while DNA Ligase III was recruited via its BRCT domain mediated interaction with XRCC1. Selective recruitment of specialized DNA Ligases may have evolved to accommodate the particular requirements of different repair pathways and may thus enhance efficiency of DNA repair

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

Wnt signaling is boosted during intestinal regeneration by a CD44-positive feedback loop

Enhancement of Wnt signaling is fundamental for stem cell function during intestinal regeneration. Molecular modules control Wnt activity by regulating signal transduction. CD44 is such a positive regulator and a Wnt target gene. While highly expressed in intestinal crypts and used as a stem cell marker, its role during intestinal homeostasis and regeneration remains unknown. Here we propose a CD44 positive-feedback loop that boosts Wnt signal transduction, thus impacting intestinal regeneration. Excision of Cd44 in Cd44$^{fl/fl}$;VillinCreER$^{T2}$ mice reduced Wnt target gene expression in intestinal crypts and affected stem cell functionality in organoids. Although the integrity of the intestinal epithelium was conserved in mice lacking CD44, they were hypersensitive to dextran sulfate sodium, and showed more severe inflammation and delayed regeneration. We localized the molecular function of CD44 at the Wnt signalosome, and identified novel DVL/CD44 and AXIN/CD44 complexes. CD44 thus promotes optimal Wnt signaling during intestinal regeneration

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

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

“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