RNA molecular recording with an engineered RNA deaminase.

RNA deaminases are powerful tools for base editing and RNA molecular recording. However, the enzymes used in currently available RNA molecular recorders such as TRIBE, DART or STAMP have limitations due to RNA structure and sequence dependence. We designed a platform for directed evolution of RNA molecular recorders. We engineered an RNA A-to-I deaminase (an RNA adenosine base editor, rABE) that has high activity, low bias and low background. Using rABE, we present REMORA (RNA-encoded molecular recording in adenosines), wherein deamination by rABE writes a molecular record of RNA-protein interactions. By combining rABE with the C-to-U deaminase APOBEC1 and long-read RNA sequencing, we measured binding by two RNA-binding proteins on single messenger RNAs. Orthogonal RNA molecular recording of mammalian Pumilio proteins PUM1 and PUM2 shows that PUM1 competes with PUM2 for a subset of sites in cells. Furthermore, we identify transcript isoform-specific RNA-protein interactions driven by isoform changes distal to the binding site. The genetically encodable RNA deaminase rABE enables single-molecule identification of RNA-protein interactions with cell type specificity.

DDX3 depletion represses translation of mRNAs with complex 5' UTRs.

DDX3 is an RNA chaperone of the DEAD-box family that regulates translation. Ded1, the yeast ortholog of DDX3, is a global regulator of translation, whereas DDX3 is thought to preferentially affect a subset of mRNAs. However, the set of mRNAs that are regulated by DDX3 are unknown, along with the relationship between DDX3 binding and activity. Here, we use ribosome profiling, RNA-seq, and PAR-CLIP to define the set of mRNAs that are regulated by DDX3 in human cells. We find that while DDX3 binds highly expressed mRNAs, depletion of DDX3 particularly affects the translation of a small subset of the transcriptome. We further find that DDX3 binds a site on helix 16 of the human ribosomal rRNA, placing it immediately adjacent to the mRNA entry channel. Translation changes caused by depleting DDX3 levels or expressing an inactive point mutation are different, consistent with different association of these genetic variant types with disease. Taken together, this work defines the subset of the transcriptome that is responsive to DDX3 inhibition, with relevance for basic biology and disease states where DDX3 is altered.

Copper regulates rest-activity cycles through the locus coeruleus-norepinephrine system.

The unusually high demand for metals in the brain, along with insufficient understanding of how their dysregulation contributes to neurological diseases, motivates the study of how inorganic chemistry influences neural circuitry. We now report that the transition metal copper is essential for regulating rest-activity cycles and arousal. Copper imaging and gene expression analysis in zebrafish identifies the locus coeruleus-norepinephrine (LC-NE) system, a vertebrate-specific neuromodulatory circuit critical for regulating sleep, arousal, attention, memory and emotion, as a copper-enriched unit with high levels of copper transporters CTR1 and ATP7A and the copper enzyme dopamine ß-hydroxylase (DBH) that produces NE. Copper deficiency induced by genetic disruption of ATP7A, which loads copper into DBH, lowers NE levels and hinders LC function as manifested by disruption in rest-activity modulation. Moreover, LC dysfunction caused by copper deficiency from ATP7A disruption can be rescued by restoring synaptic levels of NE, establishing a molecular CTR1-ATP7A-DBH-NE axis for copper-dependent LC function.

Trends in Socioeconomic Inequalities in Full Vaccination Coverage among Vietnamese Children aged 12-23 Months, 2000-2014: Evidence for Mitigating Disparities in Vaccination.

There has been no report on the situation of socioeconomic inequalities in the full vaccination coverage among Vietnamese children. This study aims to assess the trends and changes in the socioeconomic inequalities in the full vaccination coverage among Vietnamese children aged 12-23 months from 2000 to 2014. Data were drawn from Multiple Indicator Cluster Surveys (2000, 2006, 2011, and 2014). Concentration index (CCI) and concentration curve (CC) were applied to quantify the degree of the socioeconomic inequalities in full immunization coverage. The prevalence of children fully receiving recommended vaccines was significantly improved during 2000-2014, yet, was still not being covered. The total CCI of full vaccination coverage gradually decreased from 2000 to 2014 (CCI: from 0.241 to 0.009). The CC increasingly became close to the equality line through the survey period, indicating the increasingly narrow gap in child full immunization amongst the poor and the rich. Vietnam witnessed a sharp decrease in socioeconomic inequality in the full vaccination coverage for over a decade. The next policies towards children from vulnerable populations (ethnic minority groups, living in rural areas, and having a mother with low education) belonging to lower socioeconomic groups may mitigate socioeconomic inequalities in full vaccination coverage.

Multimodal LA-ICP-MS and nanoSIMS imaging enables copper mapping within photoreceptor megamitochondria in a zebrafish model of Menkes disease.

Copper is essential for eukaryotic life, and animals must acquire this nutrient through the diet and distribute it to cells and organelles for proper function of biological targets. Indeed, mutations in the central copper exporter ATP7A contribute to a spectrum of diseases, including Menkes disease, with symptoms ranging from neurodegeneration to lax connective tissue. As such, a better understanding of the fundamental impacts of ATP7A mutations on in vivo copper distributions is of relevance to those affected by these diseases. Here we combine metal imaging and optical imaging techniques at a variety of spatial resolutions to identify tissues and structures with altered copper levels in the Calamitygw71 zebrafish model of Menkes disease. Rapid profiling of tissue slices with LA-ICP-MS identified reduced copper levels in the brain, neuroretina, and liver of Menkes fish compared to control specimens. High resolution nanoSIMS imaging of the neuroretina, combined with electron and confocal microscopies, identified the megamitochondria of photoreceptors as loci of copper accumulation in wildtype fish, with lower levels of megamitochondrial copper observed in Calamitygw71 zebrafish. Interestingly, this localized copper decrease does not result in impaired photoreceptor development or altered megamitochondrial morphology, suggesting the prioritization of copper at sufficient levels for maintaining essential mitochondrial functions. Together, these data establish the Calamitygw71 zebrafish as an optically transparent in vivo model for the study of neural copper misregulation, illuminate a role for the ATP7A copper exporter in trafficking copper to the neuroretina, and highlight the utility of combining multiple imaging techniques for studying metals in whole organism settings with spatial resolution.

Effects of operating parameters on the efficiency of liposomal encapsulation of enzymes.

Encapsulation of active proteins in the hydrophilic core of vesicular liposomes is important for developing a therapeutic protein carrier system. The efficiency of liposomal encapsulation of proteins is generally low. A better understanding of the fundamental mechanisms of encapsulation is needed to increase this efficiency. In this study we investigated the effects of operating parameters such as phospholipid concentration, buffer pH and ionic strength, protein size and surface charge, and liposome size on the enzyme encapsulation yield. Four model enzymes of different molecular weights and isoelectric points (trypsin, horseradish peroxidase, enterokinase and hyaluronidase) were encapsulated into three different sized liposomes (125 nm, 194 nm, and 314 nm in mean diameter). Relatively inert and neutral DPPC (1,2-dipalmitoyl-sn-glycero-3-phosphocholine) was used as the main phospholipid in the liposomes. Size exclusion chromatography was used to separate the enzyme-encapsulated liposomes from the free enzyme, and the encapsulation yield was determined from the peak area. The encapsulation yield was generally low ranging from ca. 5% to 20%, and did not depend much on the molecular weight of the enzyme encapsulated. Larger liposomes had higher encapsulation yields. The electrostatic interaction between the phospholipid and enzyme was the most significant parameter in determining the encapsulation yield. Thus adjusting buffer pH and ionic strength and adding charged phospholipids to the liposome preparation to impart electric charge to the lipid bilayer could significantly improve the yield. This approach can be used to optimize the liposomal encapsulation of clinically significant proteins.

Potential application of antibody-mimicking peptides identified by phage display in immuno-magnetic separation of an antigen.

Phage display was performed against human IgG (hIgG) through five rounds of 'biopanning'. Each round consisted of: (1) incubating a library of phage-displayed 12-mer peptides sequences on hIgG-coated magnetic beads, (2) washing the unbound phages, and (3) eluting the bound phages. The eluted phages were either amplified to enrich the pool of positive clones or subjected to the next round without amplification. Through ELISA, four clones (F9, D1, G5, and A10) showing specific binding affinity to hIgG were identified. Among these, F9 had the highest affinity (K(d)=6.2 nM), only one order of magnitude lower than the native anti-hIgG antibody (0.66 nM). Following the DNA sequences of the selected clones, four 12-mer peptides were chemically synthesized. Among them, D1 peptide showed the highest binding affinity to hIgG via SPR biosensor measurements. This peptide was conjugated to biofunctionalized magnetic beads, and its immuno-binding ability was compared with that of the native antibody immobilized to magnetic beads. The mol-to-mol binding efficacy of the peptide-coated magnetic beads was approximately 1000-fold lower than that of the antibody-coated magnetic beads. Our results suggest a feasibility of using antibody-mimicking peptides identified by phage display technique for immuno-magnetic separation of an antigen.