Two-Photon Imaging of Calcium in Virally Transfected Striate Cortical Neurons of Behaving Monkey

Two-photon scanning microscopy has advanced our understanding of neural signaling in non-mammalian species and mammals. Various developments are needed to perform two-photon scanning microscopy over prolonged periods in non-human primates performing a behavioral task. In striate cortex in two macaque monkeys, cortical neurons were transfected with a genetically encoded fluorescent calcium sensor, memTNXL, using AAV1 as a viral vector. By constructing an extremely rigid and stable apparatus holding both the two-photon scanning microscope and the monkey's head, single neurons were imaged at high magnification for prolonged periods with minimal motion artifacts for up to ten months. Structural images of single neurons were obtained at high magnification. Changes in calcium during visual stimulation were measured as the monkeys performed a fixation task. Overall, functional responses and orientation tuning curves were obtained in 18.8% of the 234 labeled and imaged neurons. This demonstrated that the two-photon scanning microscopy can be successfully obtained in behaving primates

Challenges of implementing nano-specific safety and safe-by-design principles in academia

Safe-by-design is an essential component for creating awareness of the potential novel risks associated with the introduction of sophisticated nanomaterials (NMs) with novel properties. SbD is also a useful tool for meeting EU policy ambitions such as the European Green Deal which includes circular economy and moving towards a zero pollution (pollution-free) environment. Unidentified risks are a growing concern with the rapid and exponential advances of nanotechnology innovation, and the increase in fundamental research on NMs and their potential applications. Therefore, addressing nano-specific safety issues early in the innovation process is vital for reducing the uncertainties of novel NMs. The challenge is that many innovators and material scientists are not toxicologist and are not aware on how to assess the safety of their innovations and novel materials. Safe-by-design is a concept that aims at reducing uncertainties and risks for humans and the environment, starting at an early phase of the innovation process and covering the whole innovation value chain, including research. This perspective tries to get a better understanding on the role of safe-by-design within engineered nanomaterial research to create awareness on the importance on assessing the safety early in research. A method was developed that integrates SbD with a set of questions to aid material scientists assess the safety of their materials (nano-specific safety aspects) and Risk Analysis and Technology Assessment (RATA). Here we present the results of a workshop for material scientists (PhD students) with limited toxicology knowledge at the Debye Institute for Nanomaterials Science (Utrecht University, The Netherlands) with the main goals to create awareness with regard to basic NM safety and to explore the possibilities for applying safe-by-design principles in academia. The approach presented here can be applied by researchers and innovators to assess the safety of NMs at an early stage of the innovation process, and this work is framed in the context of Responsible Research and Innovation using RATA

The tumbleweed: towards a synthetic protein motor

Biomolecular motors have inspired the design and construction of artificial nanoscale motors and machines based on nucleic acids, small molecules, and inorganic nanostructures. However, the high degree of sophistication and efficiency of biomolecular motors, as well as their specific biological function, derives from the complexity afforded by protein building blocks. Here, we discuss a novel bottom-up approach to understanding biological motors by considering the construction of synthetic protein motors. Specifically, we present a design for a synthetic protein motor that moves along a linear track, dubbed the “Tumbleweed.” This concept uses three discrete ligand-dependent DNA-binding domains to perform cyclically ligand-gated, rectified diffusion along a synthesized DNA molecule. Here we describe how de novo peptide design and molecular biology could be used to produce the Tumbleweed, and we explore the fundamental motor operation of such a design using numerical simulations. The construction of this and more sophisticated protein motors is an exciting challenge that is likely to enhance our understanding of the structure-function relationship in biological motors.Biomolecular motors have inspired the design and construction of artificial nanoscale motors and machines based on nucleic acids, small molecules, and inorganic nanostructures. However, the high degree of sophistication and efficiency of biomolecular motors, as well as their specific biological function, derives from the complexity afforded by protein building blocks. Here, we discuss a novel bottom-up approach to understanding biological motors by considering the construction of synthetic protein motors. Specifically, we present a design for a synthetic protein motor that moves along a linear track, dubbed the “Tumbleweed.” This concept uses three discrete ligand-dependent DNA-binding domains to perform cyclically ligand-gated, rectified diffusion along a synthesized DNA molecule. Here we describe how de novo peptide design and molecular biology could be used to produce the Tumbleweed, and we explore the fundamental motor operation of such a design using numerical simulations. The construction of this and more sophisticated protein motors is an exciting challenge that is likely to enhance our understanding of the structure-function relationship in biological motors