Improved delivery of Cas9 protein/gRNA complexes using lipofectamine CRISPRMAX

Objectives

To identify the best lipid nanoparticles for delivery of purified Cas9 protein and gRNA complexes (Cas9 RNPs) into mammalian cells and to establish the optimal conditions for transfection.

Results

Using a systematic approach, we screened 60 transfection reagents using six commonly-used mammalian cell lines and identified a novel transfection reagent (named Lipofectamine CRISPRMAX). Based on statistical analysis, the genome modification efficiencies in Lipofectamine CRISPRMAX-transfected cell lines were 40 or 15 % higher than those in Lipofectamine 3000 or RNAiMAX-transfected cell lines, respectively. Upon optimization of transfection conditions, we observed 85, 75 or 55 % genome editing efficiencies in HEK293FT cells, mouse ES cells, or human iPSCs, respectively. Furthermore, we were able to co-deliver donor DNA with Cas9 RNPs into a disrupted EmGFP stable cell line, resulting in the generation of up to 17 % EmGFP-positive cells.

Conclusion

Lipofectamine CRISPRMAX was characterized as the best lipid nanoparticles for the delivery of Cas9 RNPs into a variety of mammalian cell lines, including mouse ES cells and iPSCs.

     

Transracial adoption: white American adoptive mothers’ constructions of social capital in raising their adopted children

In the field of adoption and child welfare, there is ongoing debate and discussion about how white adoptive parents in transracial families construct personal and social relationships and networks to promote cultural belonging and identity development in their children. In spite of this however, there is, to date, no research study that has sought to apply the notion of social capital to understand transracial adoptive families. With its exploration of how white American adoptive mothers construct social capital in raising their transracially adopted children, this paper seeks to contribute to the literature on social capital and families in general, and social capital and transracial families in particular. By drawing upon a qualitative study involving white adoptive mothers’ discursive practices, and multiplex constructions of intersectionality, the paper seeks to offer rich theoretical and empirical insights into social capital and transracial adoption to contribute to the literature in this area.     

A new strain ofRhodotorula rubra isolated from yogurt

Summary A new strain ofRhodotorula rubra has been isolated from yogurt which shows promise as a source for pigment and protein feed for aquacultured animals. The pigment was extracted by rupturing the cells with the French press followed by extraction with acetone and purification of the acetone extract using petroleum ether and cold 10% NaCl. The absorption spectrum indicated that the pigment was a carotenoid, the chemistry of which was examined using nuclear magnetic resonance, mass spectroscopy and resonance Raman spectroscopy. A reverse-phase HPLC equipped with octadecylsilylated (ODS) silica column showed nearly 80-times more pigment production under similar cultural conditions thatPhaffia rhodozyma. The isolate grows optimally at 20°C when grown on a variety of media. Its morphology has been studied using transmission electron microscopy, scanning electron microscopy and phase contrast microscopy. From the results of the API system, the isolate was identified asRhodotorula rubra.     

Global phylogeography of the avian malaria pathogen Plasmodium relictum based on MSP1 allelic diversity

Knowing the genetic variation that occurs in pathogen populations and how it is distributed across geographical areas is essential to understand parasite epidemiology, local patterns of virulence, and evolution of host‐resistance. In addition, it is important to identify populations of pathogens that are evolutionarily independent and thus ‘free’ to adapt to hosts and environments. Here, we investigated genetic variation in the globally distributed, highly invasive avian malaria parasite Plasmodium relictum, which has several distinctive mitochondrial haplotyps (cyt b lineages, SGS1, GRW11 and GRW4). The phylogeography of P. relictum was accessed using the highly variable nuclear gene merozoite surface protein 1 (MSP1), a gene linked to the invasion biology of the parasite. We show that the lineage GRW4 is evolutionarily independent of GRW11 and SGS1 whereas GRW11 and SGS1 share MSP1 alleles and thus suggesting the presence of two distinct species (GRW4 versus SGS1 and GRW11). Further, there were significant differences in the global distribution of MSP1 alleles with differences between GRW4 alleles in the New and the Old World. For SGS1, a lineage formerly believed to have both tropical and temperate transmission, there were clear differences in MSP1 alleles transmitted in tropical Africa compared to the temperate regions of Europe and Asia. Further, we highlight the occurrence of multiple MSP1 alleles in GRW4 isolates from the Hawaiian Islands, where the parasite has contributed to declines and extinctions of endemic forest birds since it was introduced. This study stresses the importance of multiple independent loci for understanding patterns of transmission and evolutionary independence across avian malaria parasites.     

High Resolution Mapping of Modafinil Induced Changes in Glutamate Level in Rat Brain

Modafinil is marketed in the United States for the treatment of narcolepsy and daytime somnolence due to shift-work or sleep apnea. Investigations of this drug in the treatment of cocaine and nicotine dependence in addition to disorders of executive function are also underway. Modafinil has been known to increase glutamate levels in rat brain models. Proton magnetic resonance spectroscopy (¹HMRS) has been commonly used to detect the glutamate (Glu) changes in vivo. In this study, we used a recently described glutamate chemical exchange saturation transfer (GluCEST) imaging technique to measure Modafinil induced regional Glu changes in rat brain and compared the results with Glu concentration measured by single voxel ¹HMRS. No increases in either GluCEST maps or ¹HMRS were observed after Modafinil injection over a period of 5 hours. However, a significant increase in GluCEST (19±4.4%) was observed 24 hours post Modafinil administration, which is consistent with results from previous biochemical studies. This change was not consistently seen with ¹HMRS. GluCEST mapping allows regional cerebral Glu changes to be measured and may provide a useful clinical biomarker of Modafinil effects for the management of patients with sleep disorders and addiction.     

Regioselective synthesis of isoxazole–mercaptobenzimidazole hybrids and their in vivo analgesic and anti-inflammatory activity studies

Regioselective synthesis of isoxazole–mercaptobenzimidazole hybrids and their efficiency in in vivo analgesic and anti-inflammatory activity was described. A comparison of structure–activity relationship for there compounds was also emphasized.     

MicroCT Analysis of Symphyseal Ontogeny in Archaeolemur

Previous analyses of symphyseal fusion in the extinct Malagasy lemur Archaeolemur identified several functional characteristics of joint morphology that vary postnatally (Ravosa and Simons in American Journal of Physical Anthropology 95:63–76, 1994). To complement that study, we used an imaging technique (microCT) that provides novel data on ontogenetic and local variation in biomineralization along the mandibular symphysis before complete ossification among adult Archaeolemur. Our sample of unfused symphyses comprised juveniles from the 2 earliest postnatal dental ages examined previously. We imaged each specimen (ca.18 μm volume elements) with slices parallel to the coronal plane, i.e., orthogonal to the joint articular surface. In ≤5 labiolingually equidistant joint sites, we collected 40 contiguous slices (18-μm intervals). Each of the 5 joint sites is represented by 1 slice, with biomineralization values sampled at 5 equidistant points along the articular surface and at 3 external cortical bone points. Our analysis of Archaeolemur indicates the presence of ontogenetic increases in bone mineral density accompanying increases in joint size and the number and distribution of symphyseal rugosities. Such postnatal changes are particularly marked for the middle of the joint presumed to lie adjacent to a degrading fibrocartilage pad. In Archaeolemur, labial regions of the symphysis ossify earlier and are likewise more biomineralized. Ontogenetic increases in symphyseal biomineralization, overall size, and fusion are consistent with elevated masticatory stresses owing to the postweaning shift to adult-like feeding behaviors. However, the labiolingual pattern of fusion and biomineralization in Archaeolemur appears related more to constraints on synostosis owing to the lingually located vascular supply characteristic of mammalian symphyses.     

Reactions of Nitric Oxide with Nitronyl Nitroxides and Oxygen: Prediction of Nitrite and Nitrate Formation by Kinetic Simulation

Nitric oxide reacts with nitronyl nitroxides (NNO) to form imino nitroxides (INO) and this transformation can be monitored using electron spin resonance spectroscopy. Recently, Akaike et al., reported that NNO such as 2-phenyl-4,4,5,5-tetramethylimidazoline-3-oxide-1-oxyl (PTIO) and its derivatives (e.g., carboxy-PTIO) react with nitric oxide (·NO) in a 1:1 stoichiometry forming 2-phenyl-4,4,5,5-tetra-methylimidazoline-1-oxyl (PTI) or the respective product (e.g., carboxy-PTI) together with nitrite and nitrate (Akaike et al., Biochemistry 32, 827-832, 1993). In this paper, we reevaluate their results and show that the stoichiometry of the reaction between PTIO and ·NO is 0.63 ± 0.06:1.0. The reason for this discrepancy is due to an erroneous assumption by Akaike et al., that the stoichiometry for the reaction between ·NO and O₂ is 2:1 in aqueous solution. If the data reported by Akaike et al., were recalculated using a 4:1 stoichiometry established for the aqueous oxidation of ·NO, the reaction between ·NO and PTIO would give a stoichiometry of 0.5:1.0 in closer agreement with our data. We propose a mechanism for the reaction between PTIO and ·NO in aqueous solution. This mechanism predicts that the stoichiometry between carboxy-PTIO and ·NO is dependent on the rate of generation of ·NO and is 1:1 only at low rates of ·NO generation (i.e., 10^-13 M/s). However the stoichiometry approaches 0.5:1.0 at higher rates of ·NO production or when it is added as a bolus. The ratio between nitrite and nitrate also varies as a function of the rate of generation of ·NO. The model agrees with previous experimental observations that the aqueous oxidation of ·NO in air saturated solutions will exclusively form nitrite and predicts that ·NO will only generate substantial amounts of nitrate if it is released at a rate less than 10^-17 M/s. This may have important consequences in cellular systems where the concentration of ·NO is typically measured from nitrite production.