Neuron-microelectrode junction induced by an engineered synapse organizer.

The conventional microelectrodes for recording neuronal activities do not have innate selectivity to cell type, which is one of the critical limitations for the detailed analysis of neuronal circuits. In this study, we engineered a downsized variant of the artificial synapse organizer based on neurexin1ß and a peptide-tag, fabricated gold microelectrodes functionalized with the receptor for the organizer, and performed validation experiments in primary cultured neurons. Successful inductions of synapse-like junctions were detected at the sites of contact between neurons expressing the engineered synapse organizer and functionalized microelectrodes, but not in the negative control experiment in which the electrode functionalization was omitted. Such a molecularly inducible neuron-microelectrode junction could be the basis for the next-generation electrophysiological technique enabling cell type-selective recording.

Development of artificial synapse organizers liganded with a peptide tag for molecularly inducible neuron-microelectrode interface.

It has been proposed that cell-type-specific bioelectronic interfaces for neuronal circuits could be established by utilizing the function of synapse organizers. For this purpose, using neurexin-1ß and a peptide tag, we engineered compact synapse organizers that do not interact with the naturally occurring receptors but induce presynaptic differentiation upon contact with nanobody-decorated objects in cultured mammalian and chick forebrain neurons. In chick neurons, the engineered organizer exerted synaptogenesis typically in ∼4 h after the contact, even under an air atmosphere at room temperature, thereby providing a useful cellular model for establishing the molecularly inducible neuron-microelectrode interface.

Epitope-tag-mediated synaptogenic activity in an engineered neurexin-1ß lacking the binding interface with neuroligin-1.

Clustering of neurexin-1ß occurs through the formation of a trans-cellular complex with neuroligin-1, which promotes the generation of presynapse. While the extracellular region of neurexin-1ß functions to constitute the heterophilic binding interface with neuroligin-1, it has remained unclear whether the region could also play any key role in exerting the intracellular signaling for presynaptic differentiation. In this study, we generated neurexin-1ß lacking the binding site to neuroligin-1 and with a FLAG epitope at the N-terminus, and examined its activity in cultured neurons. The engineered protein still exhibited robust synaptogenic activities upon the epitope-mediated clustering, indicating that the region for complex formation and that for transmitting presynapse differentiation signals are structurally independent of each other. Using a fluorescence protein as an epitope, synaptogenesis was also induced by a gene-codable nanobody. The finding opens possibilities of neurexin-1ß as a platform for developing various molecular tools which may allow, for example, precise modifications of neural wirings under genetic control.

APOBEC3A and 3C decrease human papillomavirus 16 pseudovirion infectivity.

Apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like (APOBEC) proteins are cellular DNA/RNA-editing enzymes that play pivotal roles in the innate immune response to viral infection. APOBEC3 (A3) proteins were reported to hypermutate the genome of human papillomavirus 16 (HPV16), the causative agent of cervical cancer. However, hypermutation did not affect viral DNA maintenance, leaving the exact role of A3 against HPV infection elusive. Here we examine whether A3 proteins affect the virion assembly using an HPV16 pseudovirion (PsV) production system, in which PsVs are assembled from its capsid proteins L1/L2 encapsidating a reporter plasmid in 293FT cells. We found that co-expression of A3A or A3C in 293FT cells greatly reduced the infectivity of PsV. The reduced infectivity of PsV assembled in the presence of A3A, but not A3C, was attributed to the decreased copy number of the encapsidated reporter plasmid. On the other hand, A3C, but not A3A, efficiently bound to L1 in co-immunoprecipitation assays, which suggests that this physical interaction may lead to reduced infectivity of PsV assembled in the presence of A3C. These results provide mechanistic insights into A3s' inhibitory effects on the assembly phase of the HPV16 virion.

Reverse pH-dependent fluorescence protein visualizes pattern of interfacial proton dynamics during hydrogen evolution reaction.

Reverse pH-dependent fluorescent protein, including dKeima, is a type of fluorescent protein in which the chromophore protonation state depends inversely on external pH. The dependence is maintained even when immobilized at the metal-solution interface. But, interestingly, its responses to the hydrogen evolution reaction (HER) at the interface are not reversed: HER rises the pH of the solution around the cathode, but, highly active HER induces chromophore deprotonation regardless of the reverse pH dependence, reflecting an interface-specific deprotonation effect by HER. Here, we exploit this phenomenon to perform scanning-less, real-time visualization of interfacial proton dynamics during HER at a wide field of view. By using dKeima, the HER-driven deprotonation effect was well discriminated from the solution pH effect. In the electrodes of composite structures with a catalyst, dKeima visualized keen dependence of the proton depletion pattern on the electrode configuration. In addition, propagations of optical signals were observed, which seemingly reflect long-range proton hopping confined to the metal-solution interface. Thus, reverse pH-dependent fluorescent proteins provide a unique tool for spatiotemporal analysis of interfacial proton dynamics, which is expected to contribute to a better understanding of the HER process and ultimately to the safe and efficient production of molecular hydrogen.

Detection of hypermutated human papillomavirus type 16 genome by Next-Generation Sequencing.

Human papillomavirus type 16 (HPV16) is a major cause of cervical cancer. We previously demonstrated that C-to-T and G-to-A hypermutations accumulated in the HPV16 genome by APOBEC3 expression in vitro. To investigate in vivo characteristics of hypermutation, differential DNA denaturation-PCR (3D-PCR) was performed using three clinical specimens obtained from HPV16-positive cervical dysplasia, and detected hypermutation from two out of three specimens. One sample accumulating hypermutations in both E2 and the long control region (LCR) was further subjected to Next-Generation Sequencing, revealing that hypermutations spread across the LCR and all early genes. Notably, hypermutation was more frequently observed in the LCR, which contains a viral replication origin and the early promoter. APOBEC3 expressed abundantly in an HPV16-positive cervix, suggesting that single-stranded DNA exposed during viral replication and transcription may be efficient targets for deamination. The results further strengthen a role of APOBEC3 in introducing HPV16 hypermutation in vivo.