Abstract

Chromosome condensation is essential for the faithful transmission of genetic information to daughter cells during cell division.However, the chromosome condensation process has been still poorly understood. Here I introduce two factors, chromosome scaffold structure and divalent cations, are required for chromosome condensation. Chromosome scaffold was found in chromosome arms as an axial structure of chromosome and it has important role in chromosome construction. The scaffold structure has been observed as a single tick axis in a single chromatid. Using super-resolution microscopy and electron microscopy, I revealed that the scaffold structure mainly consists of two thin axes. It will generate stiffness and elasticity to chromosome. Although chromosome structure was severely changed without chromosome scaffold structure, chromosome condensation itself can be achieved by another factor, divalent cations. One of divalent cations, Ca²⁺ depletion caused defects in proper mitotic progression and chromosome condensation after the breakdown of the nuclear envelope. Fluorescence lifetime imaging microscopy-Förster resonance energy transfer and electron microscopy demonstrated that chromosome condensation is influenced by Ca²⁺. Ca²⁺ is also required for stabilization of kinetochore microtubules by loading CENP-F to kinetochore. Thus, combination of several imaging techniques is highly advantageous to understand chromosome structure.

Abstract

Development and application of bio-based and bio-inspired technologies to (i) screen for, identify and quantify molecules of interest; (ii)  profile and characterize molecular activities/events; and (iii) help elucidate pathophysiological mechanisms is major field _with applications in various fields including biomedical, environmental, energy, food and agriculture. In this talk, we aim to introduce development and exploitation of biosensor technology. Specifically, we will highlight the fundamental chemical principles intrinsic in biological interactions and their exploitation in development of this technology.The exploited molecular interactions are largely driven by natural affinities between biological molecules such as substrate-enzyme, antibody-antigen, receptor-ligand, and Watson-Crick base-pairing. The figure shows an example of a biosensor for detection of Alzheimer’s disease biomarker (amyloid beta peptide) fabricated using antibody sensing platform in conjunction with electrochemical impedance spectroscopy.Advances in science and technology has allowed the inclusion of artificially synthesised molecules that mimic biomolecules of interests, such as peptide nucleic acid (PNA) and molecularly imprinted polymers (MIPs).

Sustainable development goals (SDGs) were adopted in 2015 at the UN General Assembly as the 2030 Development agenda. They are a set of 17 inter-related goalswith overarching elements: peace, prosperity, health, planet and partnerships. In this talk, besides discussing biosensor technology and some applications, we will highlight the challenges that we face in this field and the prevailing gaps between different economies in the world. We will provide specific examples of areas that this technology could be useful in the less developed economies, and hopefully open a discussion point for future collaboration efforts.

Keywords: Bio-Inspired technologies, Nature, Sustainable development, Biosensor, Sustainable development goals (SDGs).

Abstract

We devised a simple and rapid method to analyze fidelity of reverse transcriptase (RT) using next-generation sequencing (NGS). The method comprises a cDNA synthesis reaction from standard RNA with a primer containing a tag of 14 randomized bases and the RT to be tested, PCR using high-fidelity DNA polymerase, and NGS. By comparing the sequence of each read with the reference sequence, mutations were identified. The mutation can be identified to be due to an error introduced by either cDNA synthesis, PCR, or NGS based on whether the sequence reads with the same tag contain the same mutation or not. The error rates in cDNA synthesis with thermostable variant of Moloney mouse leukemia virus (MMLV) RT, MM4 (E286R/E302K/L435R/D524A), or the recently developed 16-tuple variant of family B DNA polymerase with RT activity, RTX, from Thermococcus kodakarensis, were 0.75−1.0 × 10^-4 errors/base, while that in the reaction with the wild-type human immunodeficiency virus type 1 (HIV-1) RT was 2.6 × 10^-4 errors/base. Overall, our method could precisely evaluate the fidelity of various RTs with different reaction conditions in a high-throughput manner without the use of expensive optics and troublesome adaptor ligation.

We next optimized the conditions so that one NGS run could handle cDNA products from multiple cDNA synthesis reactions performed at different conditions. This was achieved by using a primer containing not only the tag of 14 randomized bases to label each cDNA molecule but also a tag of five bases to label each reaction condition. With this method, we quantitated the error rates of 44 cDNA synthesis reactions by retroviral RTs or genetically engineered DNA polymerases with RT activity under different conditions. The results indicated that high concentrations of MgCl₂, Mn(OCOCH₃)₂, and dNTP decrease the fidelity and that these effects are more pronounced in reactions using RT from human immunodeficiency virus type 1.

Keywords:  cDNA, DNA polymerase, error rate, NGS, reverse transcriptase