Conditional Guide RNAs: Programmable Conditional Regulation of CRISPR/Cas Function in Bacterial and Mammalian Cells via Dynamic RNA Nanotechnology

A guide RNA (gRNA) directs the function of a CRISPR protein effector to a target gene of choice, providing a versatile programmable platform for engineering diverse modes of synthetic regulation (edit, silence, induce, bind). However, the fact that gRNAs are constitutively active places limitations on the ability to confine gRNA activity to a desired location and time. To achieve programmable control over the scope of gRNA activity, here we apply principles from dynamic RNA nanotechnology to engineer conditional guide RNAs (cgRNAs) whose activity is dependent on the presence or absence of an RNA trigger. These cgRNAs are programmable at two levels, with the trigger-binding sequence controlling the scope of the effector activity and the target-binding sequence determining the subject of the effector activity. We demonstrate molecular mechanisms for both constitutively active cgRNAs that are conditionally inactivated by an RNA trigger (ON → OFF logic) and constitutively inactive cgRNAs that are conditionally activated by an RNA trigger (OFF → ON logic). For each mechanism, automated sequence design is performed using the reaction pathway designer within NUPACK to design an orthogonal library of three cgRNAs that respond to different RNA triggers. In E. coli expressing cgRNAs, triggers, and silencing dCas9 as the protein effector, we observe a median conditional response of ≈4-fold for an ON → OFF “terminator switch” mechanism, ≈15-fold for an ON → OFF “splinted switch” mechanism, and ≈3-fold for an OFF → ON “toehold switch” mechanism; the median crosstalk within each cgRNA/trigger library is <2%, ≈2%, and ≈20% for the three mechanisms. To test the portability of cgRNA mechanisms prototyped in bacteria to mammalian cells, as well as to test generalizability to different effector functions, we implemented the terminator switch in HEK 293T cells expressing inducing dCas9 as the protein effector, observing a median ON → OFF conditional response of ≈4-fold with median crosstalk of ≈30% for three orthogonal cgRNA/trigger pairs. By providing programmable control over both the scope and target of protein effector function, cgRNA regulators offer a promising platform for synthetic biology

Harmonically confined, semiflexible polymer in a channel: response to a stretching force and spatial distribution of the endpoints

We consider an inextensible, semiflexible polymer or worm-like chain which is confined in the transverse direction by a parabolic potential and subject to a longitudinal force at the ends, so that the polymer is stretched out and backfolding is negligible. Simple analytic expressions for the partition function, valid in this regime, are obtained for chains of arbitrary length with a variety of boundary conditions at the ends. The spatial distribution of the end points or radial distribution function is also analyzed.Comment: 14 pages including figure

As estratégias de desenvolvimento socioeconômico da Guiné-Bissau (1973-2015)

TCC (graduação) - Universidade Federal de Santa Catarina. Centro Sócio-Econômico. Economia.Inicialmente, o trabalho apresenta um panorama histórico da situação sócio político e socioeconômico da Guiné-Bissau, depois, um debate conceitual entre os teóricos sobre a problemática do desenvolvimento socioeconômico. Logo, por um olhar histórico analítico do que realizou nas políticas econômicas implementadas no país ao longo da sua história. Em conclusão, constatou-se adoção das estratégias incoerentes com a estrutura socioeconômica do país. No caso concreto do processo da industrialização, foram realizados muitos investimentos para industrialização avançada do setor moderno sem estrutura básica para sua sustentabilidade. O resultado desse processo foi o completo fracasso do processo de industrialização e das estratégias de desenvolvimento

Articular Cartilage Repair of the Knee in Children and Adolescents

Articular cartilage predominantly serves a biomechanical function, which begins in utero and further develops during growth and locomotion. With regard to its 2-tissue structure (chondrocytes and matrix), the regenerative potential of hyaline cartilage defects is limited. Children and adolescents are increasingly suffering from articular cartilage and osteochondral deficiencies. Traumatic incidents often result in damage to the joint surfaces, while repetitive microtrauma may cause osteochondritis dissecans. When compared with their adult counterparts, children and adolescents have a greater capacity to regenerate articular cartilage defects. Even so, articular cartilage injuries in this age group may predispose them to premature osteoarthritis. Consequently, surgery is indicated in young patients when conservative measures fail. The operative techniques for articular cartilage injuries traditionally performed in adults may be performed in children, although an individualized approach must be tailored according to patient and defect characteristics. Clear guidelines for defect dimension–associated techniques have not been reported. Knee joint dimensions must be considered and correlated with respect to the cartilage defect size. Particular attention must be given to the subchondral bone, which is frequently affected in children and adolescents. Articular cartilage repair techniques appear to be safe in this cohort of patients, and no differences in complication rates have been reported when compared with adult patients. Particularly, autologous chondrocyte implantation has good biological potential, especially for large-diameter joint surface defects

Conditional Dicer Substrate Formation via Shape and Sequence Transduction with Small Conditional RNAs

RNA interference (RNAi) mediated by small interfering RNAs (siRNAs) enables knockdown of a gene of choice, executing the logical operation: silence gene Y. The fact that the siRNA is constitutively active is a significant limitation, making it difficult to confine knockdown to a specific locus and time. To achieve spatiotemporal control over silencing, we seek to engineer small conditional RNAs (scRNAs) that mediate ‘conditional RNAi’ corresponding to the logical operation: if gene X is transcribed, silence independent gene Y. By appropriately selecting gene X, knockdown of gene Y could then be restricted in a tissue- and time-specific manner. To implement the logic of conditional RNAi, our approach is to engineer scRNAs that, upon binding to mRNA ‘detection target’ X, perform shape and sequence transduction to form a Dicer substrate targeting independent mRNA ‘silencing target’ Y, with subsequent Dicer processing yielding an siRNA targeting mRNA Y for destruction. Toward this end, here we design and experimentally validate diverse scRNA mechanisms for conditional Dicer substrate formation. Test tube studies demonstrate strong OFF/ON conditional response, with at least an order of magnitude increase in Dicer substrate production in the presence of the cognate mRNA detection target. By appropriately dimensioning and/or chemically modifying the scRNAs, only the product of signal transduction, and not the reactants or intermediates, is efficiently processed by Dicer, yielding siRNAs. These mechanism studies explore diverse design principles for engineering scRNA signal transduction cascades including reactant stability vs metastability, catalytic vs noncatalytic transduction, pre- vs post-transcriptional transduction, reactant and product molecularity, and modes of molecular self-assembly and disassembly

Conditional Guide RNAs: Programmable Conditional Regulation of CRISPR/Cas Function in Bacterial and Mammalian Cells via Dynamic RNA Nanotechnology

A guide RNA (gRNA) directs the function of a CRISPR protein effector to a target gene of choice, providing a versatile programmable platform for engineering diverse modes of synthetic regulation (edit, silence, induce, bind). However, the fact that gRNAs are constitutively active places limitations on the ability to confine gRNA activity to a desired location and time. To achieve programmable control over the scope of gRNA activity, here we apply principles from dynamic RNA nanotechnology to engineer conditional guide RNAs (cgRNAs) whose activity is dependent on the presence or absence of an RNA trigger. These cgRNAs are programmable at two levels, with the trigger-binding sequence controlling the scope of the effector activity and the target-binding sequence determining the subject of the effector activity. We demonstrate molecular mechanisms for both constitutively active cgRNAs that are conditionally inactivated by an RNA trigger (ON → OFF logic) and constitutively inactive cgRNAs that are conditionally activated by an RNA trigger (OFF → ON logic). For each mechanism, automated sequence design is performed using the reaction pathway designer within NUPACK to design an orthogonal library of three cgRNAs that respond to different RNA triggers. In E. coli expressing cgRNAs, triggers, and silencing dCas9 as the protein effector, we observe a median conditional response of ≈4-fold for an ON → OFF “terminator switch” mechanism, ≈15-fold for an ON → OFF “splinted switch” mechanism, and ≈3-fold for an OFF → ON “toehold switch” mechanism; the median crosstalk within each cgRNA/trigger library is <2%, ≈2%, and ≈20% for the three mechanisms. To test the portability of cgRNA mechanisms prototyped in bacteria to mammalian cells, as well as to test generalizability to different effector functions, we implemented the terminator switch in HEK 293T cells expressing inducing dCas9 as the protein effector, observing a median ON → OFF conditional response of ≈4-fold with median crosstalk of ≈30% for three orthogonal cgRNA/trigger pairs. By providing programmable control over both the scope and target of protein effector function, cgRNA regulators offer a promising platform for synthetic biology

Reprint of "Decision support models for supplier development: Systematic literature review and research agenda"

The continuing trend towards sourcing components and semi-finished goods for less vertically integrated manufacturing systems globally leads to a dramatic increase in supply options for companies. To ensure that companies benefit from the potentials global sourcing offers, supplier-buyer relationships need to be managed efficiently. Due to the decreasing share of value-adding activities provided in-house, suppliers are more and more considered as an essential contributor to the buying company's competitive position. Consequently, to realize and sustain competitive advantages, companies try to establish institutionalized long-term relationships to their most important suppliers and to actively improve the productivity and performance of their supplier base. To support supplier development in practice, researchers have developed decision support models that provide assistance in selecting and implementing suitable supplier development activities. The aim of this paper is to provide a comprehensive and systematic overview of decision support models for supplier development and to develop a research agenda that helps to identify promising areas for future research in this area. First, typical applications for supplier development as well as potential development measures that can be adopted to improve the performance of suppliers are identified. Secondly, a systematic literature review with a focus on decision support models for supplier development is conducted. Based on the analysis of the literature, we define a research agenda that synthesizes key trends and promising research opportunities and thus highlight areas where more decision support models are needed to foster supplier development initiatives in practice

Dendritic cell-mediated vaccination relies on interleukin-4 receptor signaling to avoid tissue damage after Leishmania major infection of BALB/c mice

Prevention of tissue damages at the site of Leishmania major inoculation can be achieved if the BALB/c mice are systemically given L. major antigen (LmAg)-loaded bone marrow-derived dendritic cells (DC) that had been exposed to CpG-containing oligodeoxynucleotides (CpG ODN). As previous studies allowed establishing that interleukin-4 (IL-4) is involved in the redirection of the immune response towards a type 1 profile, we were interested in further exploring the role of IL-4. Thus, wild-type (wt) BALB/c mice or DC-specific IL-4 receptor alpha (IL-4Rα)-deficient (CD11ccreIL-4Rα−/lox) BALB/c mice were given either wt or IL-4Rα-deficient LmAg-loaded bone marrow-derived DC exposed or not to CpG ODN prior to inoculation of 2×105 stationary-phase L. major promastigotes into the BALB/c footpad. The results provide evidence that IL4/IL-4Rα-mediated signaling in the vaccinating DC is required to prevent tissue damage at the site of L. major inoculation, as properly conditioned wt DC but not IL-4Rα-deficient DC were able to confer resistance. Furthermore, uncontrolled L. major population size expansion was observed in the footpad and the footpad draining lymph nodes of CD11ccreIL-4Rα−/lox mice immunized with CpG ODN-exposed LmAg-loaded IL-4Rα-deficient DC, indicating the influence of IL-4Rα-mediated signaling in host DC to control parasite replication. In addition, no footpad damage occurred in BALB/c mice that were systemically immunized with LmAg-loaded wt DC doubly exposed to CpG ODN and recombinant IL-4. We discuss these findings and suggest that the IL4/IL4Rα signaling pathway could be a key pathway to trigger when designing vaccines aimed to prevent damaging processes in tissues hosting intracellular microorganisms