Expansion microscopy of zebrafish for neuroscience and developmental biology studies.

Expansion microscopy (ExM) allows scalable imaging of preserved 3D biological specimens with nanoscale resolution on fast diffraction-limited microscopes. Here, we explore the utility of ExM in the larval and embryonic zebrafish, an important model organism for the study of neuroscience and development. Regarding neuroscience, we found that ExM enabled the tracing of fine processes of radial glia, which are not resolvable with diffraction-limited microscopy. ExM further resolved putative synaptic connections, as well as molecular differences between densely packed synapses. Finally, ExM could resolve subsynaptic protein organization, such as ring-like structures composed of glycine receptors. Regarding development, we used ExM to characterize the shapes of nuclear invaginations and channels, and to visualize cytoskeletal proteins nearby. We detected nuclear invagination channels at late prophase and telophase, potentially suggesting roles for such channels in cell division. Thus, ExM of the larval and embryonic zebrafish may enable systematic studies of how molecular components are configured in multiple contexts of interest to neuroscience and developmental biology.

Programmable RNA-binding protein composed of repeats of a single modular unit.

The ability to monitor and perturb RNAs in living cells would benefit greatly from a modular protein architecture that targets unmodified RNA sequences in a programmable way. We report that the RNA-binding protein PumHD (Pumilio homology domain), which has been widely used in native and modified form for targeting RNA, can be engineered to yield a set of four canonical protein modules, each of which targets one RNA base. These modules (which we call Pumby, for Pumilio-based assembly) can be concatenated in chains of varying composition and length, to bind desired target RNAs. The specificity of such Pumby-RNA interactions was high, with undetectable binding of a Pumby chain to RNA sequences that bear three or more mismatches from the target sequence. We validate that the Pumby architecture can perform RNA-directed protein assembly and enhancement of translation of RNAs. We further demonstrate a new use of such RNA-binding proteins, measurement of RNA translation in living cells. Pumby may prove useful for many applications in the measurement, manipulation, and biotechnological utilization of unmodified RNAs in intact cells and systems.

Co-occurrence, possible origin, and health-risk assessment of arsenic and fluoride in drinking water sources in Mexico: Geographical data visualization.

Arsenic and fluoride in drinking water present a significant challenge to public health worldwide. In this study, we analyze the results of one of the largest surveys of drinking water quality in Mexico: 14,058 samples from 3951 sites, collected between January and December 2017. We use these data to identify the distribution and possible origin of arsenic and fluoride in drinking water throughout the country, and to estimate the associated health burden. The highest concentrations appear in alluvial aquifers in arid northern Mexico, where high-silica volcanic rock likely releases both arsenic and fluoride to the groundwater. We find fluoride contamination to be significantly correlated with aridity (Pearson correlation = -0.45, p = 0.0105), and also find a significant difference in fluoride concentrations between arid and humid states (Welch's t-test, p = 0.004). We estimate population exposure by assigning to each town in Mexico the average concentration of any sampling sites within 5 km. Our results show that 56% of the Mexican population lives within 5 km of a sampling site, 3.05 million people are exposed to fluoride above the reference dosage of 0.06 mg/(kg ∗ day), 8.81 million people are exposed to arsenic above the limit of 10 µg/L, and an additional 13,070 lifetime cases of cancer are expected from this arsenic exposure alone. This burden of disease is concentrated in the arid states of north-central Mexico.

Engineering genetic circuit interactions within and between synthetic minimal cells.

Genetic circuits and reaction cascades are of great importance for synthetic biology, biochemistry and bioengineering. An open question is how to maximize the modularity of their design to enable the integration of different reaction networks and to optimize their scalability and flexibility. One option is encapsulation within liposomes, which enables chemical reactions to proceed in well-isolated environments. Here we adapt liposome encapsulation to enable the modular, controlled compartmentalization of genetic circuits and cascades. We demonstrate that it is possible to engineer genetic circuit-containing synthetic minimal cells (synells) to contain multiple-part genetic cascades, and that these cascades can be controlled by external signals as well as inter-liposomal communication without crosstalk. We also show that liposomes that contain different cascades can be fused in a controlled way so that the products of incompatible reactions can be brought together. Synells thus enable a more modular creation of synthetic biology cascades, an essential step towards their ultimate programmability.