Photoactivation of LOV domains with chemiluminescence.

Optogenetics has opened new possibilities in the remote control of diverse cellular functions with high spatiotemporal precision using light. However, delivering light to optically non-transparent systems remains a challenge. Here, we describe the photoactivation of light-oxygen-voltage-sensing domains (LOV domains) with in situ generated light from a chemiluminescence reaction between luminol and H2O2. This activation is possible due to the spectral overlap between the blue chemiluminescence emission and the absorption bands of the flavin chromophore in LOV domains. All four LOV domain proteins with diverse backgrounds and structures (iLID, BcLOV4, nMagHigh/pMagHigh, and VVDHigh) were photoactivated by chemiluminescence as demonstrated using a bead aggregation assay. The photoactivation with chemiluminescence required a critical light-output below which the LOV domains reversed back to their dark state with protein characteristic kinetics. Furthermore, spatially confined chemiluminescence produced inside giant unilamellar vesicles (GUVs) was able to photoactivate proteins both on the membrane and in solution, leading to the recruitment of the corresponding proteins to the GUV membrane. Finally, we showed that reactive oxygen species produced by neutrophil like cells can be converted into sufficient chemiluminescence to recruit the photoswitchable protein BcLOV4-mCherry from solution to the cell membrane. The findings highlight the utility of chemiluminescence as an endogenous light source for optogenetic applications, offering new possibilities for studying cellular processes in optically non-transparent systems.

Reversible photoregulation of cell-cell adhesions with opto-E-cadherin.

E-cadherin-based cell-cell adhesions are dynamically and locally regulated in many essential processes, including embryogenesis, wound healing and tissue organization, with dysregulation manifesting as tumorigenesis and metastasis. However, the lack of tools that would provide control of the high spatiotemporal precision observed with E-cadherin adhesions hampers investigation of the underlying mechanisms. Here, we present an optogenetic tool, opto-E-cadherin, that allows reversible control of E-cadherin-mediated cell-cell adhesions with blue light. With opto-E-cadherin, functionally essential calcium binding is photoregulated such that cells expressing opto-E-cadherin at their surface adhere to each other in the dark but not upon illumination. Consequently, opto-E-cadherin provides remote control over multicellular aggregation, E-cadherin-associated intracellular signalling and F-actin organization in 2D and 3D cell cultures. Opto-E-cadherin also allows switching of multicellular behaviour between single and collective cell migration, as well as of cell invasiveness in vitro and in vivo. Overall, opto-E-cadherin is a powerful optogenetic tool capable of controlling cell-cell adhesions at the molecular, cellular and behavioural level that opens up perspectives for the study of dynamics and spatiotemporal control of E-cadherin in biological processes.

Rational Design of Thermoresponsive Microgel Templates with Polydopamine Surface Coating for Microtissue Applications.

Functional microgels provide a versatile basis for synthetic in vitro platforms as alternatives to animal experiments. The tuning of the physical, chemical, and biological properties of synthetic microgels can be achieved by blending suitable polymers and formulating them such to reflect the heterogenous and complex nature of biological tissues. Based on this premise, this paper introduces the development of volume-switchable core-shell microgels as 3D templates to enable cell growth for microtissue applications, using a systematic approach to tune the microgel properties based on a deep conceptual and practical understanding. Microscopic microgel design, such as the tailoring of the microgel size and spherical shape, is achieved by droplet-based microfluidics, while on a nanoscopic scale, a thermoresponsive polymer basis, poly(N-isopropylacrylamide) (PNIPAAm), is used to provide the microgel volume switchability. Since PNIPAAm has only limited cell-growth promoting properties, the cell adhesion on the microgel is further improved by surface modification with polydopamine, which only slightly affects the microgel properties, thereby simplifying the system. To further tune the microgel thermoresponsiveness, different amounts of N-hydroxyethylacrylamide are incorporated into the PNIPAAm network. In a final step, cell growth on the microgel surface is investigated, both at a single microgel platform and in spheroidal cell structures.

Spatiotemporal Control Over Multicellular Migration Using Green Light Reversible Cell-Cell Interactions.

The regulation of cell-cell adhesions in space and time plays a crucial role in cell biology, especially in the coordination of multicellular behavior. Therefore, tools that allow for the modulation of cell-cell interactions with high precision are of great interest to a better understanding of their roles and building tissue-like structures. Herein, the green light-responsive protein CarH is expressed at the plasma membrane of cells as an artificial cell adhesion receptor, so that upon addition of its cofactor vitamin B12 specific cell-cell interactions form and lead to cell clustering in a concentration-dependent manner. Upon green light illumination, the CarH based cell-cell interactions disassemble and allow for their reversion with high spatiotemporal control. Moreover, these artificial cell-cell interactions impact cell migration, as observed in a wound-healing assay. When the cells interact with each other in the presence of vitamin B12 in the dark, the cells form on a solid front and migrate collectively; however, under green light illumination, individual cells migrate randomly out of the monolayer. Overall, the possibility of precisely controlling cell-cell interactions and regulating multicellular behavior is a potential pathway to gaining more insight into cell-cell interactions in biological processes.

Orthogonal Blue and Red Light Controlled Cell-Cell Adhesions Enable Sorting-out in Multicellular Structures.

The self-assembly of different cell types into multicellular structures and their organization into spatiotemporally controlled patterns are both challenging and extremely powerful to understand how cells function within tissues and for bottom-up tissue engineering. Here, we not only independently control the self-assembly of two cell types into multicellular architectures with blue and red light, but also achieve their self-sorting into distinct assemblies. This required developing two cell types that form selective and homophilic cell-cell interactions either under blue or red light using photoswitchable proteins as artificial adhesion molecules. The interactions were individually triggerable with different colors of light, reversible in the dark, and provide noninvasive and temporal control over the cell-cell adhesions. In mixtures of the two cells, each cell type self-assembled independently upon orthogonal photoactivation, and cells sorted out into separate assemblies based on specific self-recognition. These self-sorted multicellular architectures provide us with a powerful tool for producing tissue-like structures from multiple cell types and investigate principles that govern them.

Description of Plasmodium falciparum infections in central Gabon demonstrating high parasite densities among symptomatic adolescents and adults.

BACKGROUND: Malaria remains a public health issue, particularly in sub-Saharan Africa with special features of seriousness in young children and pregnant women. Adolescents and adults are reported to have acquired a semi-immune status and, therefore, present with low parasitaemia. Children are understood to present with a much higher parasitaemia and severe malaria. It is a concern that effective malaria control programmes targeting young children may lead to a delay in the acquisition of acquired immunity and, therefore, causing a shift in the epidemiology of malaria. Prevalence and parasitaemia were explored in adolescents and adults with Plasmodium falciparum infections compared to young children in the area of Lambaréné, Gabon as an indicator for semi-immunity. METHODS: A cross-sectional study was conducted at the Centre de Recherches Médicales de Lambaréné (CERMEL) during a 6-month period in 2018. Symptomatic patients, of all ages were screened for malaria at health facilities in Lambaréné and Fougamou and their respective surrounding villages in the central region of Gabon. Plasmodium falciparum infections were determined either by rapid diagnostic test (RDT) or by microscopy. Descriptive analysis of data on parasite densities, anaemia, and fever are presented. RESULTS: 1589 individuals screened were included in this analysis, including 731 (46%) adolescents and adults. Out of 1377 assessed, the proportion of P. falciparum positive RDTs was high among adolescents (68%) and adults (44%), compared to young children (55%) and school children (72%). Out of 274 participants assessed for malaria by microscopy, 45 (16%) had a parasite count above 10,000/µl of which 9 (20%) were adults. CONCLUSION: This study shows a high rate of P. falciparum infections in adolescents and adults associated with high-level parasitaemia similar to that of young children. Adolescents and adults seem to be an at-risk population, suggesting that malaria programmes should consider adolescents and adults during the implementation of malaria prevention and case management programmes with continuous care, since they also act as reservoirs for P. falciparum.

DNA recovery from archived RDTs for genetic characterization of Plasmodium falciparum in a routine setting in Lambaréné, Gabon.

BACKGROUND: Rapid diagnostic tests (RDTs) have been described as a source of genetic material to analyse malaria parasites in proof-of-concept studies. The increasing use of RDTs (e.g., in focal or mass screening and treatment campaigns) makes this approach particularly attractive for large-scale investigations of parasite populations. In this study, the complexity of Plasmodium falciparum infections, parasite load and chloroquine resistance transporter gene mutations were investigated in DNA samples extracted from positive RDTs, obtained in a routine setting and archived at ambient temperature. METHODS: A total of 669 archived RDTs collected from malaria cases in urban, semi-urban and rural areas of central Gabon were used for P. falciparum DNA extraction. Performance of RDTs as a source of DNA for PCR was determined using: (i) amplification of a single copy merozoite surface protein 1 (msp1) gene followed by highly sensitive and automated capillary electrophoresis; (ii) genotyping of the pfcrt gene locus 72-76 using haplotype-specific-probe-based real-time PCR to characterize chloroquine resistance; and, (iii) real-time PCR targeting 18S genes to detect and quantify Plasmodium parasites. RESULTS: Out of the 669 archived RDTs, amplification of P. falciparum nucleic materials had a success rate of 97% for 18S real-time PCR, and 88% for the msp1 gene. The multiplicity of infections (MOI) of the whole population was 2.6 (95% CI 2.5-2.8). The highest number of alleles detected in one infection was 11. The MOI decreased with increasing age (ß = - 0.0046, p = 0.02) and residence in Lambaréné was associated with smaller MOIs (p < 0.001). The overall prevalence of mutations associated with chloroquine resistance was 78.5% and was not associated with age. In Lambaréné, prevalence of chloroquine resistance was lower compared to rural Moyen-Ogooué (ß = - 0.809, p-value = 0.011). CONCLUSION: RDT is a reliable source of DNA for P. falciparum detection and genotyping assays. Furthermore, the increasing use of RDTs allows them to be an alternative source of DNA for large-scale genetic epidemiological studies. Parasite populations in the study area are highly diverse and prevalence of chloroquine-resistant P. falciparum remains high, especially in rural areas.

MitoCeption: Transferring Isolated Human MSC Mitochondria to Glioblastoma Stem Cells.

Mitochondria play a central role for cell metabolism, energy production and control of apoptosis. Inadequate mitochondrial function has been found responsible for very diverse diseases, ranging from neurological pathologies to cancer. Interestingly, mitochondria have recently been shown to display the capacity to be transferred between cell types, notably from human mesenchymal stem cells (MSC) to cancer cells in coculture conditions, with metabolic and functional consequences for the mitochondria recipient cells, further enhancing the current interest for the biological properties of these organelles. Evaluating the effects of the transferred MSC mitochondria in the target cells is of primary importance to understand the biological outcome of such cell-cell interactions. The MitoCeption protocol described here allows the transfer of the mitochondria isolated beforehand from the donor cells to the target cells, using MSC mitochondria and glioblastoma stem cells (GSC) as a model system. This protocol has previously been used to transfer mitochondria, isolated from MSCs, to adherent MDA-MB-231 cancer cells. This mitochondria transfer protocol is adapted here for GSCs that present the specific particularity of growing as neurospheres in vitro. The transfer of the isolated mitochondria can be followed by fluorescence-activated cell sorting (FACS) and confocal imaging using mitochondria vital dyes. The use of mitochondria donor and target cells with distinct haplotypes (SNPs) also allows detection of the transferred mitochondria based on the concentration of their circular mitochondrial DNA (mtDNA) in the target cells. Once the protocol has been validated with these criteria, the cells harboring the transferred mitochondria can be further analyzed to determine the effects of the exogenous mitochondria on biological properties such as cell metabolism, plasticity, proliferation and response to therapy.