An in silico method to assess antibody fragment polyreactivity

Antibodies are essential biological research tools and important therapeutic agents, but some exhibit non-specific binding to off-target proteins and other biomolecules. Such polyreactive antibodies compromise screening pipelines, lead to incorrect and irreproducible experimental results, and are generally intractable for clinical development. We designed a set of experiments using a diverse naive synthetic camelid antibody fragment ('nanobody') library to enable machine learning models to accurately assess polyreactivity from protein sequence (AUC > 0.8). Moreover, our models provide quantitative scoring metrics that predict the effect of amino acid substitutions on polyreactivity. We experimentally tested our model's performance on three independent nanobody scaffolds, where over 90% of predicted substitutions successfully reduced polyreactivity. Importantly, the model allowed us to diminish the polyreactivity of an angiotensin II type I receptor antagonist nanobody, without compromising its pharmacological properties. We provide a companion web-server that provides a straightforward means of predicting polyreactivity and polyreactivity-reducing mutations for any given nanobody sequence.

The Current Status and Future Direction of Clinical Research in Japan From a Regulatory Perspective

In Japan, a law called the Clinical Trials Act went into being effective on April 1, 2018, and clinical research on human subjects conducted in Japan has been undergone major changes. Those other than clinical trials for marketing approval of drugs or medical devices are broadly classified into “specific clinical trials” and others, and regulations have been tightened for each. As a result, clinical interventional study was drastically reduced, and observational clinical study increased. For the observational clinical study, the two previous ethical guidelines were merged into the “Ethical Guidelines for Medical and Biological Research Involving Human Subjects,” which was enacted in March 2021. The observational clinical study is now subjected to these ethical guidelines. In addition, changes are planned for the Act on the Protection of Personal Information, which greatly affects data collection in clinical research. Clinical research in Japan must be conducted appropriately while adapting to these various changes in the external environment and legal framework. Adapting to these changes is not an easy task, as it requires increased financial and human resources for all stakeholders.

HyU: Hybrid Unmixing for longitudinal in vivo imaging of low signal to noise fluorescence

The expanded application of fluorescence imaging in biomedical and biological research towards more complex systems and geometries requires tools that can analyze a multitude of components at widely varying time- and length-scales. The major challenge in such complex imaging experiments is to cleanly separate multiple fluorescent labels with overlapping spectra from one another and background autofluorescence, without perturbing the sample with high levels of light. Thus, there is a requirement for efficient and robust analysis tools capable of quantitatively separating these signals. In response, we have combined multispectral fluorescence microscopy with hyperspectral phasors and linear unmixing to create Hybrid Unmixing (HyU). Here we demonstrate its capabilities in the dynamic imaging of multiple fluorescent labels in live, developing zebrafish embryos. HyU is more sensitive to low light levels of fluorescence compared to conventional linear unmixing approaches, permitting better multiplexed volumetric imaging over time, with less bleaching. HyU can also simultaneously image both bright exogenous and dim endogenous labels because of its high dynamic range. This allows studies of cellular behaviors, tagged components, and cell metabolism within the same specimen, offering a powerful window into the orchestrated complexity of biological systems.

Generative adversarial network enables rapid and robust fluorescence lifetime image analysis in live cells

Fluorescence lifetime imaging microscopy (FLIM) is a powerful tool to quantify molecular compositions and study molecular states in complex cellular environment as the lifetime readings are not biased by fluorophore concentration or excitation power. However, the current methods to generate FLIM images are either computationally intensive or unreliable when the number of photons acquired at each pixel is low. Here we introduce a new deep learning-based method termed flimGANE (fluorescence lifetime imaging based on Generative Adversarial Network Estimation) that can rapidly generate accurate and high-quality FLIM images even in the photon-starved conditions. We demonstrated our model is up to 2,800 times faster than the gold standard time-domain maximum likelihood estimation (TD_MLE) and that flimGANE provides a more accurate analysis of low-photon-count histograms in barcode identification, cellular structure visualization, Förster resonance energy transfer characterization, and metabolic state analysis in live cells. With its advantages in speed and reliability, flimGANE is particularly useful in fundamental biological research and clinical applications, where high-speed analysis is critical.

A Novel Biomimetic Nanocomposite Exhibiting Petal Wetting Phenomenon: Fabrication and Experimental Investigations

A functional composite material that simultaneously exhibits hydrophobicity and water droplet adhesion has monumental potential in controlling fluid flow, studying phase separation, and biological research. This article reports the fabrication of a petal wetting biomimetic Boron Nitride Nanotubes (BNNTs) -Polydimethylsiloxane (PDMS) nanocomposite achieved by drop casting. The petal effect was investigated by non-destructive techniques. The nanotubes were synthesized by chemical vapor deposition at 1150 °C and were characterized by X-ray diffraction, scanning electron microscopy, and high-resolution transmission electron microscopy. The mean diameter of the nanotubes was found to be 70 nm. The nanocomposites had BNNT fillers ranging from 0.5 wt. % to 2 wt. %. Water contact angles for pure PDMS polymer was 94.7° and for the 2 wt. % BNNT-PDMS nanocomposite was 132.4°. The petal wetting nanocomposite displayed a characteristic trait of high contact angle hysteresis. The surface roughness parameters of the nanocomposites were determined by atomic force microscopy. Laser scanning confocal microscopy aided in analyzing the droplet penetration and in observing the trapped air between the water droplet and the nanocomposite surface. Based on surface observations, roughness parameters, and the extent of droplet penetration by the surface, we shed light on the Cassie impregnating wetting regime followed by the biomimetic nanocomposite. Such a surface would be beneficial in the study of the embryogenesis of cells and aid in moisture collection.

Exploration of Methanomethylophilus alvus pyrrolysyl-tRNA synthetase activity in yeast

Archaeal pyrrolysyl-tRNA synthetases (PylRSs) have been used to genetically encode over 200 distinct noncanonical amino acids (ncAAs) in proteins in E. coli and mammalian cells. This vastly expands the range of chemical functionality accessible within proteins produced in these organisms. Despite these clear successes, explorations of PylRS function in yeast remains limited. In this work, we demonstrate that the Methanomethylophilus alvus PylRS (MaPylRS) and its cognate tRNACUA support the incorporation of ncAAs into proteins produced in S. cerevisiae using stop codon suppression methodologies. Additionally, we prepared three MaPylRS mutants originally engineered in E. coli and determined that all three were translationally active with one or more ncAAs, although with low efficiencies of ncAA incorporation in comparison to the parent MaPylRS. Alongside MaPylRS variants, we evaluated the translational activity of previously reported Methanosarcina mazei, Methanosarcina barkeri, and chimeric M. mazei and M. barkeri PylRSs. Using the yeast strain RJY100, and pairing these aaRSs with the M. barkeri tRNACUA, we did not observe any detectable stop codon suppression activity under the same conditions that produced moderately efficient ncAA incorporation with MaPylRS. The addition of MaPylRS to the orthogonal translation machinery toolkit in yeast potentially opens the door to hundreds of ncAAs that have not previously been genetically encodable using other aminoacyl-tRNA synthetase/tRNA pairs. Extending the scope of ncAA incorporation in yeast could powerfully advance chemical and biological research for applications ranging from basic biological discovery to enzyme engineering and therapeutic protein lead discovery.

Systems biology–the transformative approach to integrate sciences across disciplines

Life science is the study of living organisms, including bacteria, plants, and animals. Given the importance of biology, chemistry, and bioinformatics, we anticipate that this chapter may contribute to a better understanding of the interdisciplinary connections in life science. Research in applied biological sciences has changed the paradigm of basic and applied research. Biology is the study of life and living organisms, whereas science is a dynamic subject that as a result of constant research, new fields are constantly emerging. Some fields come and go, whereas others develop into new, well-recognized entities. Chemistry is the study of composition of matter and its properties, how the substances merge or separate and also how substances interact with energy. Advances in biology and chemistry provide another means to understand the biological system using many interdisciplinary approaches. Bioinformatics is a multidisciplinary or rather transdisciplinary field that encourages the use of computer tools and methodologies for qualitative and quantitative analysis. There are many instances where two fields, biology and chemistry have intersection. In this chapter, we explain how current knowledge in biology, chemistry, and bioinformatics, as well as its various interdisciplinary domains are merged into life sciences and its applications in biological research.

CLARITY and Light-Sheet microscopy sample preparation in application to human cerebral organoids

Cerebral organoids are three-dimensional cell-culture systems that represent a unique experimental model reconstructing early events of human neurogenesis in vitro in health and various pathologies. The most commonly used approach to studying the morphological parameters of organoids is immunohistochemical analysis; therefore, the three-dimensional cytoarchitecture of organoids, such as neural networks or asymmetric internal organization, is difficult to reconstruct using routine approaches. Immunohistochemical analysis of biological objects is a universal method in biological research. One of the key stages of this method is the production of cryo- or paraffin serial sections of samples, which is a very laborious and time-consuming process. In addition, slices represent only a tiny part of the object under study; three-dimensional reconstruction from the obtained serial images is an extremely complex process and often requires expensive special programs for image processing. Unfortunately, staining and microscopic examination of samples are difficult due to their low permeability and a high level of autofluorescence. Tissue cleaning technologies combined with Light-Sheet microscopy allows these challenges to be overcome. CLARITY is one of the tissue preparation techniques that makes it possible to obtain opaque biological objects transparent while maintaining the integrity of their internal structures. This method is based on a special sample preparation, during which lipids are removed from cells and replaced with hydrogel compounds such as acrylamide, while proteins and nucleic acids remain intact. CLARITY provides researchers with a unique opportunity to study three-dimensional biological structures while preserving their internal organization, including whole animals or embryos, individual organs and artificially grown organoids, in particular cerebral organoids. This protocol summarizes an optimization of CLARITY conditions for human brain organoids and the preparation of Light-Sheet microscopy samples.

Generation of bright monomeric red fluorescent proteins via computational design of enhanced chromophore packing

Red fluorescent proteins (RFPs) have found widespread application in chemical and biological research due to their longer emission wavelengths. Here, we use computational protein design to increase the quantum yield...