Memory, imagination and the human spirit

Welker, Michael | Memory studies (v.9 / no.3 / pp.341-347 / 20161750-6980)

Learning from economic theory, the article proposes to differentiate and relate (episodic) micro-memory and micro-imagination and different types of macro-memory and macr

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Learning from economic theory, the article proposes to differentiate and relate (episodic) micro-memory and micro-imagination and different types of macro-memory and macro-imagination. Drawing on several decades of discourse in historical and cultural studies (from Halbwachs to Le #x0301;vi-Strauss and to A. and J. Assmann), it differentiates between communicative, cultural, collective and canonical memories. With reference to basic processes of moral communication and a more complex understanding of the human spirit than the one offered in the long tradition of post-Aristotelian thought, it sketches a route towards relating micro- and macro-memories and normative imaginations.

Chloride channel-3 promotes tumor metastasis by regulating membrane ruffling and is associated with poor survival.

Xu, Bin, Jin, Xiaobao, Min, Ling, Li, Qin, Deng, Lulu, Wu, Hui, Lin, Guixian, Chen, Lixin, Zhang, Haifeng, Li, Chunmei, Wang, Liwei, Zhu, Jiayong, Wang, Weizhang, Chu, Fujiang, Shen, Juan, Li, Hongzhi, Mao, Jianwen | Oncotarget (v.6 / no.4 / pp.2434-2450 / 2015)

The chloride channel-3 (ClC-3) protein is known to be a component of Cl- channels involved in cell volume regulation or acidification of intracellular vesicles. Here, we

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The chloride channel-3 (ClC-3) protein is known to be a component of Cl- channels involved in cell volume regulation or acidification of intracellular vesicles. Here, we report that ClC-3 was highly expressed in the cytoplasm of metastatic carcinomatous cells and accelerated cell migration in vitro and tumor metastasis in vivo. High-grade expression of cytoplasmic ClC-3 predicted poor survival in cancer patients. We found that independent of its volume-activated Cl- channel properties, ClC-3 was able to promote cell membrane ruffling, required for tumor metastasis. ClC-3 mediated membrane ruffling by regulating keratin 18 phosphorylation to control β1 Integrin recycling. Therefore, cytoplasmic ClC-3 plays an active and key role in tumor metastasis and may be a valuable prognostic biomarker and a therapeutic target to prevent tumor spread.

Parvovirus B19 infection as a cause of acute myositis in an adult.

Cakirca, Mustafa, Karatoprak, Cumali, Ugurlu, Serdal, Zorlu, Mehmet, Kiska?, Muharrem, ?etin, G?ven | Revista Brasileira de reumatologia (v.55 / no.2 / pp.185-188 / 20150482-5004)

Parvovirus B19 infection is often asymptomatic, but clinical expressions may include transient aplastic crisis, erythema infectiosum, non-immune hydrops fetalis, and chro

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Parvovirus B19 infection is often asymptomatic, but clinical expressions may include transient aplastic crisis, erythema infectiosum, non-immune hydrops fetalis, and chronic red cell aplasia. This virus has also been associated with rheumatoid arthritis and other autoimmune connective tissue diseases; however, we could not identify any acute adult myositis case developed after a Parvovirus B19 infection in the literature. For this reason, we would like to present a rare case of acute myositis developed after Parvovirus B19 infection. In patients presenting with symptoms of fever, rash on the legs and myositis, viral infections such as Parvovirus B19 should be kept in mind.

Your mileage may vary: Managing untargeted consumers rsquo; reactions to promotions

Thompson, S. A., Gooner, R. A., Kim, A. | Journal of the Academy of Marketing Science (v.43 / no.6 / pp.713-729 / 20150092-0703)

EPA mileage estimates are not as erroneous as many people tend to believe. That conclusion is indicated by results of recent tests conducted by SAE students at California

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Enhancing Thermomechanical Properties and Heat Distortion Resistance of Poly(l-lactide) with High Crystallinity under High Cooling Rate

Yin, Hai-Yan, Wei, Xin-Feng, Bao, Rui-Ying, Dong, Quan-Xiao, Liu, Zheng-Ying, Yang, Wei, Xie, Bang-Hu, Yang, Ming-Bo | ACS sustainable chemistry et engineering (v.3 / no.4 / pp.654-661 / 2015)

In this work, a novel, effective and simple approach to largely improve the thermomechanical properties and heat distortion resistance of biodegradable poly( l -lactide

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In this work, a novel, effective and simple approach to largely improve the thermomechanical properties and heat distortion resistance of biodegradable poly( l -lactide) (PLLA) by using a new nucleating agent (NA), i.e., itself high-melting-point homocrystallites ( h PLLA crystallites) is reported. Specially, h PLLA crystallites with a melting temperature ( T m ) of 187 °C were introduced into the PLLA matrix with a lower T m , i.e., 168 °C via simply melt blending at 170 °C which is between the T m s of the two PLLAs. Nonisothermal and isothermal crystallization results reveal that h PLLA crystallite is an efficient nucleating agent for PLLA. Also, h PLLA crystallites show much more prominently promoting effect on the crystallization rate of PLLA in comparison with two widely reported NAs for PLLA, talc and stereocomplex crystallites. Most importantly, this promoting effect is still efficient at very high cooling rate, leading to a crystallinity of 39.1% at a cooling rate of 100 °C/min, which can help to obtain high-crystallinity PLLA products in conventional manufacturing processes. The optical microscopic observation reveals that the remarkable crystallization promotion can be attributed to the outstanding heterogeneous nucleation effect, as a result of both identical chemical constitution and lattice constitution between h PLLA crystallites and PLLA matrix. Further characterizations indicate that the enhancement of PLLA crystallinity by using such a new efficient NA can enhance the thermomechanical properties and heat distortion resistance of PLLA remarkably. For instance, at 80 °C (above T g of PLLA), the elastic modulus increases by 60 times from 8 to 477 MPa with the incorporation of 5 wt % h PLLA. High-melting-point crystallites of poly( l -lactide) show outstanding nucleation enhancement on low-melting-point poly( l -lactide), achieving high crystallinity under a high cooling rate. Graphic Abstract ACS Electronic Supporting Info

Car restraint policies and mileage in Singapore

Chu, S. | Transportation research Part A, Policy and practice (v.77 / pp.404-412 / 20150965-8564)

Negative externalities often surface after policies are implemented. This paper analyses how two ''hard'' Travel Demand Management (TDM) policies implemented in Singapore

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Negative externalities often surface after policies are implemented. This paper analyses how two ''hard'' Travel Demand Management (TDM) policies implemented in Singapore to target vehicle ownership and road usage may contribute to a negative externality namely excessive mileage accumulation. This has implications on resource depletion such as petrol wastage, higher CO 2 emission and losses in time and productivity. Vehicle ownership in Singapore is managed firstly via the requirement to bid for a Certificate of Entitlement (COE) which entitles the usage of local roads and secondly via the payment of an Additional Registration Fee (ARF) which is refundable between 75% and 50% to incentivise the de-registration of a vehicle before it is 10years old. Such deregistered vehicles may also be eligible for a COE refund between 0% and 80% depending on age. The COE and ARF costs are significant as they typically account for more than half the purchase price of a vehicle. Furthermore, road usage is subject to Electronic Road Pricing (ERP) fees on busy segments. A sample of over 8700 used cars is analysed to infer the effects of the non-refundable (or ''sunk'') and the ''variable'' portions of the combined cost of COE and ARF as well as the number of ERP gantries on mileage over and above traditional factors such as petrol price and engine size. The findings suggest tweaks to the TDM policies to reduce mileage and its negative implications.

Community-Based Recovery and Youth Justice

Sellers, Brian G. | Criminal justice and behavior (v.42 / no.1 / pp.58-69 / 20150093-8548)

Four well-known delinquency intervention and prevention programs remain both publicly and politically popular regardless of a large body of evidence-based research reveal

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Four well-known delinquency intervention and prevention programs remain both publicly and politically popular regardless of a large body of evidence-based research revealing their ineffectiveness in promoting a lasting desistance from youth violence and crime. Scared straight programs, Drug Abuse Resistance Education (D.A.R.E.), youth boot camps, and secure large-scale, custodial juvenile correctional facilities overemphasize offender #x201C;risk management and maintenance #x201D; as opposed to individual, group-based, and/or collective well-being. This article will identify the values that these current and dominant community-centered youth justice initiatives reflect, and it will explain how these values further (or forestall) offender desistance. Viable, evidence-based alternatives consistent with the value orientation of therapeutic and restorative programming will also be evaluated. The article concludes by examining the efficacy of this alternative normative agenda to foster successful desistance from juvenile delinquency and crime.

Single molecule microscopy and spectroscopy: concluding remarks

van Hulst, Niek F. | Faraday discussions (v.184 / pp.475-484 / 20151359-6640)

Chemistry is all about molecules: control, synthesis, interaction and reaction of molecules. All too easily on a blackboard, one draws molecules, their structures and dyn

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Chemistry is all about molecules: control, synthesis, interaction and reaction of molecules. All too easily on a blackboard, one draws molecules, their structures and dynamics, to create an insightful picture. The dream is to see these molecules in reality. This is exactly what ldquo;Single Molecule Detection rdquo; provides: a look at molecules in action at ambient conditions; a breakthrough technology in chemistry, physics and biology. Within the realms of the Royal Society of Chemistry, the Faraday Discussion on ldquo;Single Molecule Microscopy and Spectroscopy rdquo; was a very appropriate topic for presentation, deliberation and debate. Undoubtedly, the Faraday Discussions have a splendid reputation in stimulating scientific debates along the traditions set by Michael Faraday. Interestingly, back in the 1830's, Faraday himself pursued an experiment that led to the idea that atoms in a compound were joined by an electrical component. He placed two opposite electrodes in a solution of water containing a dissolved compound, and observed that one of the elements of the compound accumulated on one electrode, while the other was deposited on the opposite electrode. Although Faraday was deeply opposed to atomism, he had to recognize that electrical forces were responsible for the joining of atoms. Probably a direct view on the atoms or molecules in his experiment would have convinced him. As such, Michael Faraday might have liked the gathering at Burlington House in September 2015 (Fig. 1). Surely, with the questioning eyes of his bust on the 1 st floor corridor, the non-believer Michael Faraday has incited each passer-by to enter into discussion and search for deeper answers at the level of single molecules. In these concluding remarks, highlights of the presented papers and discussions are summarized, complemented by a conclusion on future perspectives. The Royal Society of Chemistry at Burlington House in London. The entrance and the stained glass window on the stairway towards the first floor corridor where one finds the bronze bust representing Michael Faraday, protagonist of the early-day lively scientific discussions, which have inspired the ldquo;Faraday Discussions rdquo;.

Cultural diversity and mental health

Gopalkrishnan, Narayan, Babacan, Hurriyet | Australasian psychiatry : bulletin of the Royal Australian and New Zealand College of Psychiatrists (v.23 / no.6 / pp.6-8 / 20151039-8562)

Objective: Cultural diversity and its impact on mental health has become an increasingly important issue in a globalised world where the interactions between cultures co

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Objective: Cultural diversity and its impact on mental health has become an increasingly important issue in a globalised world where the interactions between cultures continue to grow exponentially. This paper presents critical areas in which culture impacts on mental health, such as how health and illness are perceived, coping styles, treatment-seeking patterns, impacts of history, racism, bias and stereotyping, gender, family, stigma and discrimination. Conclusions: While cultural differences provide a number of challenges to mental health policy and practice they also provide a number of opportunities to work in unique and effective ways towards positive mental health. Ethno-specific approaches to mental health that incorporate traditional and community-based systems can provide new avenues for working with culturally diverse populations.

Chloride concentrations in human hepatic cytosol and mitochondria are a function of age

Jahn, Stephan C., Rowland-Faux, Laura, Stacpoole, Peter W., James, Margaret O. | Biochemical and biophysical research communications (v.459 / no.3 / pp.463-468 / 20150006-291x)

Abstract We recently reported that, in a concentration-dependent manner, chloride protects hepatic glutathione transferase zeta 1 from inactivation by dichloroacetate, a

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Abstract We recently reported that, in a concentration-dependent manner, chloride protects hepatic glutathione transferase zeta 1 from inactivation by dichloroacetate, an investigational drug used in treating various acquired and congenital metabolic diseases. Despite the importance of chloride ions in normal physiology, and decades of study of chloride transport across membranes, the literature lacks information on chloride concentrations in animal tissues other than blood. In this study we measured chloride concentrations in human liver samples from male and female donors aged 1 day to 84 years (n?=?97). Because glutathione transferase zeta 1 is present in cytosol and, to a lesser extent, in mitochondria, we measured chloride in these fractions by high-performance liquid chromatography analysis following conversion of the free chloride to pentafluorobenzylchloride. We found that chloride concentration decreased with age in hepatic cytosol but increased in liver mitochondria. In addition, chloride concentrations in cytosol, (105.2?±?62.4?mM; range: 24.7 ndash;365.7?mM) were strikingly higher than those in mitochondria (4.2?±?3.8?mM; range 0.9 ndash;22.2?mM). These results suggest a possible explanation for clinical observations seen in patients treated with dichloroacetate, whereby children metabolize the drug more rapidly than adults following repeated doses, and also provide information that may influence our understanding of normal liver physiology. Highlights This study is the first large scale measurement of hepatic chloride in any species. The chloride concentration is much higher in human hepatic cytosol than in the mitochondria. Chloride concentration decreases with age in the cytosol while increasing in the mitochondria. Graphical abstract [DISPLAY OMISSION]

Chloride ingress in cracked and uncracked SHCC under cyclic wetting-drying exposure

Paul, S.C., van Zijl, G.P.A.G., Babafemi, A.J., Tan, M.J. | Construction building materials (v.114 / pp.232-240 / 20160950-0618)

This paper reports the performance of strain hardening cement-based composite (SHCC) containing polyvinyl alcohol (PVA) fiber under chloride exposure of both cracked and

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This paper reports the performance of strain hardening cement-based composite (SHCC) containing polyvinyl alcohol (PVA) fiber under chloride exposure of both cracked and uncracked specimens. The performance of SHCC was also compared to that of an identical mortar mixture without fibers. Three-point bending was applied to mechanically induce cracks in both reinforced SHCC and mortar specimens. Cracks were also formed in unreinforced SHCC specimens, whereas cracks were not formed in the unreinforced mortar specimens because of its brittle behavior. Thereafter, specimens were subjected to cyclic and continuous chloride attack for defined time periods. Both total and free chloride in SHCC and mortar specimens were then determined by means of X-ray florescence (XRF) and chemical analysis according to RILEM recommendations. Rapid chloride migration tests were also performed to determine the chloride migration coefficient in both SHCC and mortar. The results confirm that the mechanically-induced cracks in the SHCC specimens lead to higher chloride migration than in uncracked SHCC.

Community Health Nursing through a Global Lens.

Sarkar, Norma, Dallwig, Amber, Abbott, Patricia | Studies in health technology and informatics (v.209 / pp.135-139 / 20150926-9630)

Community Health Nursing (N456) is a required senior clinical course in the undergraduate nursing curriculum at the University of Michigan in which students learn to asse

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Community Health Nursing (N456) is a required senior clinical course in the undergraduate nursing curriculum at the University of Michigan in which students learn to assess and address the health of populations and communities. In 2012, we began our efforts to internationalize the curriculum using a globally engaged nursing education framework. Our goal is for all students to have an intercultural learning experience understanding that all students are unable to travel internationally. Therefore, this intercultural learning was implemented through a range of experiences including actual immersion, virtual activities (videoconferencing) and interventions with local vulnerable populations. Grants were obtained to provide immersion experiences in Quito, Ecuador and New Delhi, India. Several technologies were initiated with partner nursing schools in Leogane, Haiti and New Delhi, India. Weekly videoconferencing utilizing BlueJeans software and exchange of knowledge through the Knowledge Gateway facilitated intercultural exchange of knowledge and culture. Local clinical groups work with a variety of vulnerable populations. A private blog was developed for all sections to share community assessment data from local and international communities. Qualitative evaluation data was collected for local and international students to begin to assess cultural competence and student learning. Analysis of data documented increased awareness of culture and identified the many positive benefits of interaction with a global partner.

Synthesis and Characterization of Calixarene Tetraethers: An Exercise in Supramolecular Chemistry for the Undergraduate Organic Laboratory

Debbert, Stefan L., Hoh, Bradley D., Dulak, David J. | Journal of chemical education (v.93 / no.2 / pp.372-375 / 20160021-9584)

In this experiment for an introductory undergraduate organic chemistry lab, students tetraalkylate tert -butylcalix[4]arene, a bowl-shaped macrocyclic oligophenol, and

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In this experiment for an introductory undergraduate organic chemistry lab, students tetraalkylate tert -butylcalix[4]arene, a bowl-shaped macrocyclic oligophenol, and examine the supramolecular chemistry of the tetraether product by proton nuclear magnetic resonance (NMR) spectroscopy. Complexation with a sodium ion reduces the conformational mobility of the macrocycle through host ndash;guest interactions, greatly simplifying the 1 H NMR spectrum of the macrocycle and providing an excellent example of geminal coupling between the diastereotopic protons of the methylene bridges. By dealing explicitly with the organic chemistry of large molecules and host ndash;guest complementarity, this experiment provides a useful pedagogical bridge from small-molecule organic chemistry to the biochemistry of macromolecules such as enzymes. Graphic Abstract ACS Electronic Supporting Info

Imagination in human social cognition, autism, and psychotic-affective conditions

Crespi, B., Leach, E., Dinsdale, N., Mokkonen, M., Hurd, P. | Cognition (v.150 / pp.181-199 / 20160010-0277)

Complex human social cognition has evolved in concert with risks for psychiatric disorders. Recently, autism and psychotic-affective conditions (mainly schizophrenia, bip

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Complex human social cognition has evolved in concert with risks for psychiatric disorders. Recently, autism and psychotic-affective conditions (mainly schizophrenia, bipolar disorder, and depression) have been posited as psychological 'opposites' with regard to social-cognitive phenotypes. Imagination, considered as 'forming new ideas, mental images, or concepts', represents a central facet of human social evolution and cognition. Previous studies have documented reduced imagination in autism, and increased imagination in association with psychotic-affective conditions, yet these sets of findings have yet to be considered together, or evaluated in the context of the diametric model. We first review studies of the components, manifestations, and neural correlates of imagination in autism and psychotic-affective conditions. Next, we use data on dimensional autism in healthy populations to test the hypotheses that: (1) imagination represents the facet of autism that best accounts for its strongly male-biased sex ratio, and (2) higher genetic risk of schizophrenia is associated with higher imagination, in accordance with the predictions of the diametric model. The first hypothesis was supported by a systematic review and meta-analysis showing that Imagination exhibits the strongest male bias of all Autism Quotient (AQ) subscales, in non-clinical populations. The second hypothesis was supported, for males, by associations between schizophrenia genetic risk scores, derived from a set of single-nucleotide polymorphisms, and the AQ Imagination subscale. Considered together, these findings indicate that imagination, especially social imagination as embodied in the default mode human brain network, mediates risk and diametric dimensional phenotypes of autism and psychotic-affective conditions.

Chloride-promoted formation of octahedral LiNi0.5Mn1.5O4 crystal with greatly enhanced electrochemical performance

Feng, F., Liang, C., Fang, H., Yang, B., Ma, W., Dai, Y. | Ceramics international (v.42 / no.7 / pp.9038-9045 / 20160272-8842)

Two LiNi 0.5 Mn 1.5 O 4 samples are synthesized by a low-temperature solid-state method using chlorides or acetates as raw materials, and their crystal structure, s

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Two LiNi 0.5 Mn 1.5 O 4 samples are synthesized by a low-temperature solid-state method using chlorides or acetates as raw materials, and their crystal structure, size, morphology and electrochemical performance are comparatively investigated. The results show that the two samples have the same crystal structure but with different crystal size and morphology. The LiNi 0.5 Mn 1.5 O 4 prepared from chlorides shows relatively bigger crystals with a well-developed octahedral shape with {111} faces, while the LiNi 0.5 Mn 1.5 O 4 prepared from acetates displays a quasi octahedral shape whose edges and corners are not fully developed. Such a difference in crystal size and morphology leads to a big difference in their electrochemical performance, and the octahedral LiNi 0.5 Mn 1.5 O 4 prepared from chlorides has higher capacity, much better rate capability and cycling performance. Our results suggest that the crystal size and morphology difference of the two samples may be associated with the presence of unreacted chloride in the precursor derived from raw materials of chlorides, and the presence of chloride may effectively promote the formation of octahedral LiNi 0.5 Mn 1.5 O 4 crystal at low temperature via a solid-state reaction (500 o C in this work)

Senior Hunger : The Importance of Quality Assessment Tools in Determining Need

Gualtieri, Marie C., Donley, Amy M. | Journal of applied social science (v.10 / no.1 / pp.8-21 / 20161936-7244)

Food insecurity is not a new phenomenon experienced by seniors living in the United States. In a recent report by Feeding America, approximately 4.8 million Americans ove

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Food insecurity is not a new phenomenon experienced by seniors living in the United States. In a recent report by Feeding America, approximately 4.8 million Americans over the age of 60 are food insecure. The findings from this study call for major policy and funding implications. Through semistructured, face-to-face interviews, this study found how the assessment tools determining the relative need for these seniors underestimate the scope of their experiences relating to food insecurity. The assessment tool used by this particular program is based on federally recommended questions. These same questions are used by agencies across the nation that receive federal funding. This is problematic as this study shows that the current assessment tool does not properly capture some of the barriers many older Americans are facing in trying to obtain food.

A thermo-mechanical large deformation constitutive model for polymers based on material network description: Application to a semi-crystalline polyamide 66

Maurel-Pantel, A., Baquet, E., Bikard, J., Bouvard, J.L., Billon, N. | International journal of plasticity (v.67 / pp.102-126 / 20150749-6419)

A visco-hyperelastic constitutive model, based on an original approach initially developed by (Billon, 2012) and applied to amorphous rubbery polymers for a one-dimension

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A visco-hyperelastic constitutive model, based on an original approach initially developed by (Billon, 2012) and applied to amorphous rubbery polymers for a one-dimensional formalism, was extended in this study to three-dimensional constitutive equations based on a thermodynamic framework. The model was applied to a semi-crystalline polyamide polymer, PA66. The experiments included tension and shear testing coupled with synchronized digital image correlation and infrared measurements device for capturing the time, temperature, and stress state dependence, as well as the complex thermomechanical coupling exhibited by the material under large deformation. A notion of equivalent strain rate (based on the time-temperature principle superposition) was also introduced to show its capability to build master curves and therefore decrease the number of testing needed to build a material database. The model is based on the Edward Vilgis theory (1986) and accounts for chains network reorganization under external loading through the introduction of an evolution equation for the internal state variable, eta;@?, representing the degree of mobility of entanglement points. The model accounting for the equivalent strain rate notion was calibrated using master curves. The thermomechanical model agreed well with the experimental mechanical and temperature measurements under tension and shear conditions. The approach developed in this study may open a different way to model the polymer behavior.

Community forest management and REDD+

| Forest policy and economics (v.56 / pp.27-37 / 20151389-9341)

The urgent need to limit anthropogenic carbon emissions has led to the global initiative on Reducing Emissions from Deforestation and forest Degradation (REDD+). One opti

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The urgent need to limit anthropogenic carbon emissions has led to the global initiative on Reducing Emissions from Deforestation and forest Degradation (REDD+). One option to facilitate the design and implementation of REDD+ is to build on the experiences of community forest management (CFM). Despite tensions between the central objectives of REDD+ and CFM, the two policy interventions share the objective of managing forests sustainably. REDD+ projects can build on and benefit from the environmental, social, human, and institutional capital associated with existing community forest governance. Using a comparative case approach with studies from Nepal and Tanzania, we illustrate interactions between REDD+ and CFM. In Nepal, most REDD+ pilot projects have been located in community forest sites, especially in high-carbon forests. In Tanzania, REDD+ funding is being used to expand the area of forest under Participatory Forest Management. Our study also highlights how community forestry institutions may need to be modified to satisfy key REDD+ criteria. Greater institutional coordination, equitable benefit sharing mechanisms, and higher community capacity for monitoring, reporting, and verification are key areas needing change. There are significant risks, but the vast experience and significant successes of CFM can improve prospects for achieving REDD+ objectives in other less-industrialized, forested countries.

Cinematic Revolution: Cosmopolitan Alter-cinema of Pre-revolutionary Iran

Rekabtalaei, Golbarg | Iranian studies : bulletin of the Society for Iranian Cultural and Social Studies (v.48 / no.4 / pp.567-589 / 20150021-0862)

By bringing a synchronic extrapolation of certain properties of cinema and the phonograph into contact with a diachronic survey of their institutional behavior as realist

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High content analysis platform for optimization of lipid mediated CRISPR-Cas9 delivery strategies in human cells

Steyer, B., Carlson-Stevermer, J., Angenent-Mari, N., Khalil, A., Harkness, T., Saha, K. | Acta Biomaterialia: structure-property-function relationships in biomaterials (v.34 / pp.143-158 / 20161742-7061)

Non-viral gene-editing of human cells using the CRISPR-Cas9 system requires optimized delivery of multiple components. Both the Cas9 endonuclease and a single guide RNA,

초록보기원문보기 >

Non-viral gene-editing of human cells using the CRISPR-Cas9 system requires optimized delivery of multiple components. Both the Cas9 endonuclease and a single guide RNA, that defines the genomic target, need to be present and co-localized within the nucleus for efficient gene-editing to occur. This work describes a new high-throughput screening platform for the optimization of CRISPR-Cas9 delivery strategies. By exploiting high content image analysis and microcontact printed plates, multi-parametric gene-editing outcome data from hundreds to thousands of isolated cell populations can be screened simultaneously. Employing this platform, we systematically screened four commercially available cationic lipid transfection materials with a range of RNAs encoding the CRISPR-Cas9 system. Analysis of Cas9 expression and editing of a fluorescent mCherry reporter transgene within human embryonic kidney cells was monitored over several days after transfection. Design of experiments analysis enabled rigorous evaluation of delivery materials and RNA concentration conditions. The results of this analysis indicated that the concentration and identity of transfection material have significantly greater effect on gene-editing than ratio or total amount of RNA. Cell subpopulation analysis on microcontact printed plates, further revealed that low cell number and high Cas9 expression, 24h after CRISPR-Cas9 delivery, were strong predictors of gene-editing outcomes. These results suggest design principles for the development of materials and transfection strategies with lipid-based materials. This platform could be applied to rapidly optimize materials for gene-editing in a variety of cell/tissue types in order to advance genomic medicine, regenerative biology and drug discovery. Statement of Significance: CRISPR-Cas9 is a new gene-editing technology for ''genome surgery'' that is anticipated to treat genetic diseases. This technology uses multiple components of the Cas9 system to cut out disease-causing mutations in the human genome and precisely suture in therapeutic sequences. Biomaterials based delivery strategies could help transition these technologies to the clinic. The design space for materials based delivery strategies is vast and optimization is essential to ensuring the safety and efficacy of these treatments. Therefore, new methods are required to rapidly and systematically screen gene-editing efficacy in human cells. This work utilizes an innovative platform to generate and screen many formulations of synthetic biomaterials and components of the CRISPR-Cas9 system in parallel. On this platform, we watch genome surgery in action using high content image analysis. These capabilities enabled us to identify formulation parameters for Cas9-material complexes that can optimize gene-editing in a specific human cell type.