The roles of aromatic residues in the glycine receptor transmembrane domain.

BACKGROUND: Cys-loop receptors play important roles in fast neuronal signal transmission. Functional receptors are pentamers, with each subunit having an extracellular, transmembrane (TM) and intracellular domain. Each TM domain contains 4 α-helices (M1-M4) joined by loops of varying lengths. Many of the amino acid residues that constitute these α-helices are hydrophobic, and there has been particular interest in aromatic residues, especially those in M4, which have the potential to contribute to the assembly and function of the receptor via a range of interactions with nearby residues. RESULTS: Here we show that many aromatic residues in the M1, M3 and M4 α-helices of the glycine receptor are involved in the function of the receptor. The residues were explored by creating a range of mutant receptors, characterising them using two electrode voltage clamp in Xenopus oocytes, and interpreting changes in receptor parameters using currently available structural information on the open and closed states of the receptor. For 7 residues function was ablated with an Ala substitution: 3 Tyr residues at the extracellular end of M1, 2 Trp residues located towards the centers of M1 and M3, and a Phe and a Tyr residue in M4. For many of these an alternative aromatic residue restored wild-type-like function indicating the importance of the π ring. EC50s were increased with Ala substitution of 8 other aromatic residues, with those in M1 and M4 also having reduced currents, indicating a role in receptor assembly. The structure shows many potential interactions with nearby residues, especially between those that form the M1/M3/M4 interface, and we identify those that are supported by the functional data. CONCLUSION: The data reveal the importance and interactions of aromatic residues in the GlyR M1, M3 and M4 α-helices, many of which are essential for receptor function

Machine learning-guided synthesis of advanced inorganic materials

Synthesis of advanced inorganic materials with minimum number of trials is of paramount importance towards the acceleration of inorganic materials development. The enormous complexity involved in existing multi-variable synthesis methods leads to high uncertainty, numerous trials and exorbitant cost. Recently, machine learning (ML) has demonstrated tremendous potential for material research. Here, we report the application of ML to optimize and accelerate material synthesis process in two representative multi-variable systems. A classification ML model on chemical vapor deposition-grown MoS2 is established, capable of optimizing the synthesis conditions to achieve higher success rate. While a regression model is constructed on the hydrothermal-synthesized carbon quantum dots, to enhance the process-related properties such as the photoluminescence quantum yield. Progressive adaptive model is further developed, aiming to involve ML at the beginning stage of new material synthesis. Optimization of the experimental outcome with minimized number of trials can be achieved with the effective feedback loops. This work serves as proof of concept revealing the feasibility and remarkable capability of ML to facilitate the synthesis of inorganic materials, and opens up a new window for accelerating material development

Visualizing Cellular Gibberellin Levels Using the nlsGPS1 Förster Resonance Energy Transfer (FRET) Biosensor.

The phytohormone gibberellin (GA) is a small, mobile signaling molecule that plays a key role in seed germination, cellular elongation, and developmental transitions in plants. Gibberellin Perception Sensor 1 (GPS1) is the first Förster resonance energy transfer (FRET)-based biosensor that allows monitoring of cellular GA levels in vivo. By measuring a fluorescence emission ratio of nuclear localized-GPS1 (nlsGPS1), spatiotemporal mapping of endogenously and exogenously supplied GA gradients in different tissue types is feasible at a cellular scale. This protocol will describe how to image nlsGPS1 emission ratios in three example experiments: steady-state, before-and-after exogenous gibberellin A4 (GA4) treatments, and over a treatment time-course. We also provide methods to analyze nlsGPS1 emission ratios using both Fiji and a commercial three-dimensional (3-D) micrograph visualization and analysis software and explain the limitations and likely pitfalls of using nlsGPS1 to quantify gibberellin levels.European Research Council (ERC) under the European Union's Horizon 2020 research and innovation program (grant agreement n° 759282

Differential biosynthesis and cellular permeability explain longitudinal gibberellin gradients in growing roots.

Control over cell growth by mobile regulators underlies much of eukaryotic morphogenesis. In plant roots, cell division and elongation are separated into distinct longitudinal zones and both division and elongation are influenced by the growth regulatory hormone gibberellin (GA). Previously, a multicellular mathematical model predicted a GA maximum at the border of the meristematic and elongation zones. However, GA in roots was recently measured using a genetically encoded fluorescent biosensor, nlsGPS1, and found to be low in the meristematic zone grading to a maximum at the end of the elongation zone. Furthermore, the accumulation rate of exogenous GA was also found to be higher in the elongation zone. It was still unknown which biochemical activities were responsible for these mobile small molecule gradients and whether the spatiotemporal correlation between GA levels and cell length is important for root cell division and elongation patterns. Using a mathematical modeling approach in combination with high-resolution GA measurements in vivo, we now show how differentials in several biosynthetic enzyme steps contribute to the endogenous GA gradient and how differential cellular permeability contributes to an accumulation gradient of exogenous GA. We also analyzed the effects of altered GA distribution in roots and did not find significant phenotypes resulting from increased GA levels or signaling. We did find a substantial temporal delay between complementation of GA distribution and cell division and elongation phenotypes in a GA deficient mutant. Together, our results provide models of how GA gradients are directed and in turn direct root growth

Structure and phase engineering of 2D transition metal chalcogenides

As an indispensable member of the two-dimensional (2D) family as well as a perfect complement to graphene, 2D transition metal chalcogenides (TMCs) have long been the center of research attention, attributed to their large spectrum of fascinating properties along with the various crystal structures. In parallel with the discovery of new candidate materials and exploration of their unique characteristics, engineering the 2D TMCs into designated structures and phases are also of great importance for meeting the requirement for different applications. This thesis focuses on the structure and phase engineering of 2D TMCs to tune their physicochemical properties. Four strategies are adopted to tune the properties of 2D TMCs by fabricating them into desired structures, architectures, or defined phases: construction of heterostructure, alloying, phase-selective growth, and dimension tuning. Moreover, this thesis also describes and validates the feasibility and potential of introducing ML to guide the synthesis and engineering of 2D TMCs. In the first project, through the construction of MoS2-WS2 lateral heterostructures, semiconductor p-n junctions are successfully obtained, which are essential building blocks for modern electronic and optoelectronic devices. Moreover, the morphology of heterostructures can be engineered by fine-tuning the synthesis conditions. WS2 quantum well is also identified in the synthesized heterostructures, providing opportunities for studying novel optical properties and quantum confinement effects. In the second project, monolayer WTe2xS2(1-x) alloys with tunable chemical compositions and phases are fabricated using a carefully designed one-step CVD method. By controlling the synthesis condition, both semiconducting 1H and metallic 1T´ phase 2D WTe2xS2(1-x) alloys are obtained. Bandgap engineering of WTe2xS2(1-x) alloys in the 1H phase is achieved as well. Moreover, the generalizability of the proposed approach in preparing phase tunable TMCs alloys, is demonstrated for the growth of 2D WTe2xSe2(1-x) alloys. In the third project, the strategy of phase-selective growth is applied to the study of 2D Cr5Te8. Phase-tunable growth of 2D ferromagnetic Cr5Te8 is achieved via a facile CVD route. By fine-tuning the synthesis condition, both trigonal and monoclinic phase Cr5Te8 down to a few nanometers are synthesized for the first time and their ferromagnetic properties are respectively examined. Compared with the trigonal phase, monoclinic Cr5Te8 possesses a higher Curie temperature and coercivity field. Phase-dependent characteristics that existed in many 2D TMCs make phase-selective growth more useful. In the last project, the feasibility and capability of ML techniques to guide the synthesis and engineering of 2D TMCs are demonstrated. ML-guided synthesis and dimension tuning of few-layer 1T´ WTe2 are realized. An ML model with a high AUROC of 0.93 is established, to optimize the CVD synthesis conditions of few-layer 1T´ WTe2. Feature importance extracted from the model further reveals that source ratio plays a dominating role in governing the morphology of the synthesized WTe2 flakes. WTe2 nanoribbons are eventually obtained. This work suggests that ML is a powerful and efficient approach to guide the synthesis and dimension tuning of 2D materials, opening up new opportunities for boosting the diversified nanostructures derived from the 2D TMCs family.Doctor of Philosoph

Engineering and deploying FRET-based biosensors to illuminate cellular phytohormone dynamics coordinating environmental stress responses

Synthesised in plants in small quantities, phytohormones are naturally occurring chemical messengers that play critical roles in regulating plant growth and development as well as triggering responses to external stimuli. The precise regulation of phytohormone biosynthesis, catabolism and transport is crucial to maintain these messengers’ concentration, allowing different parts of the plant to communicate and coordinate responses to changing environmental conditions. Understanding the biology of phytohormones has therefore developed to be an important field of study. In this thesis, I focused on two specific hormones, Gibberellin (GA) and Salicylic acid (SA). I improved and characterised the next-generation Gibberellin Perception Sensors (GPS) based on the GPS1 in Chapter 3 and successfully designed and engineered a novel FRET-based biosensor for SA, Salicylic acid Sensor 1 (SalicS1) in Chapter 5. Using these biosensors, I examined the relationship between repatterning of the corresponding phytohormones and plant reprogramming under different stress conditions in Chapter 4 (GA) and Chapter 6 (SA). Aims of this thesis are summarised in the Figure below. These biosensors allowed the monitoring of changes in phytohormone levels with high spatial and temporal resolution and provided valuable insights into the complex interplay between phytohormones and environmental stimuli at the cellular level. While nuclear-localised GPS1 (nlsGPS1) has been found to bind bioactive GA4 with high affinity and good signal-to-noise ratio, other GPS1 biosensor properties remained to be optimised and diversified. By modulating the interaction interface between the sensory domains AtGID1C and the truncated DELLA domain of AtGAI, we have successfully increased the *in vitro* reversibility of GPS1, and GA hypersensitivity phenotypes were reduced, resulting in GPS2. In my project, GPS2 was fully characterised. I further attempted to expand the range of GPS biosensor affinities through mutagenesis and by deploying higher affinity GID1 variants from other plant species. By altering the linkers between fluorescent proteins (FPs) and binding domain, I created GPS3 with a much-improved signal to noise ratio *in vitro* which allows accurate detection of smaller changes in GA levels particularly at low concentrations, although such properties were not observed *in planta*. I used nlsGPS1 to study the relationship between GA's redistribution and abiotic stresses, including nutrient deficiency, salinity stress, high sugar stress and osmotic stress. I then focused on SA which is best known as a plant defence hormone and is also involved in increasing plant tolerance to several abiotic stresses. In this arm of my project, I developed a novel FRET based biosensor, SalicS1, to directly detect SA levels in live plants with unprecedented resolution. I screened SA receptors and their interaction partners from multiple species as ligand sensory domains. Combinations of various cyan-yellow FPs as FRET pairs and a set of linker variants connecting these four moieties generated single biosensor fusion proteins that were evaluated for the optimal SalicS1. SalicS1 response to SA was tested first *in vitro* after purification from yeast and then *in planta* in stable transgenic *Arabidopsis* lines, both in a dose dependent manner. Using more low pH tolerant FPs will allow biosensors to be more widely subcellularly targeted, particularly in the acidic environments of the vacuole and apoplasm. My preliminary evidence indicate that some FRET pairs could lead to successful low pH-tolerant biosensors. Further engineering is needed to develop high signal-to-noise ratio low pH-tolerant biosensors (SalicSLowpH and GPSLowpH) to elucidate subcellular phytohormone distribution. Nuclear localised SalicS1 (nlsSalicS1) were further used in studying the redistribution of SA levels under abiotic stresses. It revealed that SA were reduced in *Arabidopsis* seedlings roots. Other abiotic stresses, including low temperatures and salinity stress, were found to affect cellular SA levels, depending on the duration of exposure. In conclusion, I fully characterised the GPS2 and created GPS3. I also engineered a novel FRET based SA sensor, SalicS1, to allow SA levels to be monitored in cellular scale *in vivo*. I attempted to design diversified sensor variants to be targeted to acidic subcellular environments. Taken together, my thesis has engineered and applied biosensors to advance our understanding of phytohormone redistribution at a high spatiotemporal resolution under stress conditions

Multiple regions in the extracellular domain of the glycine receptor determine receptor activity.

Glycine receptors (GlyRs) are Cys-loop receptors that mediate fast synaptic inhibition in the brain stem and spinal cord. They are involved in the generation of motor rhythm, reflex circuit coordination, and sensory signal processing and therefore represent targets for therapeutic interventions. The extracellular domains (ECDs) of Cys-loop receptors typically contain many aromatic amino acids, but only those in the receptor binding pocket have been extensively studied. Here, we show that many Phe residues in the ECD that are not located in the binding pocket are also involved in GlyR function. We examined these Phe residues by creating several GlyR variants, characterizing these variants with the two-electrode voltage clamp technique in Xenopus oocytes, and interpreting changes in receptor parameters by using currently available structural information on the open and closed states of the GlyR. Substitution of six of the eight Phe residues in the ECD with Ala resulted in loss of function or significantly increased the EC50 and also altered the maximal response to the partial GlyR agonist taurine compared with glycine in those receptor variants that were functional. Substitutions with other amino acids, combined with examination of nearby residues that could potentially interact with these Phe residues, suggested interactions that could be important for GlyR function, and possibly similar interactions could contribute to the function of other members of the Cys-loop receptor family. Overall, our results suggest that many ECD regions are important for GlyR function and that these regions could inform the design of therapeutic agents targeting GlyR activity

Research data supporting "Differential biosynthesis and cellular permeability explain longitudinal gibberellin gradients in growing roots"

Datasets in support of the figures of "Differential biosynthesis and cellular permeability explain longitudinal gibberellin gradients in growing roots" publication in PNAS 2021 with first author Annalisa Rizza. See file Rizza et al.,2021 metadata confocal imaging and phenotypes .xlsx for details on biological data. The dataset contains experimental data on Arabidopsis thaliana roots and consists primarily of microscopy imaging data presented as Leica files (.lif) and root growth phenotyping data presented as Excel files (.xlsx) or TIFF (.tiff) images. Most imaging files are organised by data acquired and phenotyping files are organised by corresponding Figure number from the publication. Detailed explanation is found in the metadata excel file. Additional imaging files are presented as TIFF images (Scanned plates zip files, .tiff) and imaging and phenotyping data analysis presented as Origin Pro files (.opju). Finally, a set of modeling program files relating to the mathematical modeling described in the publication are provided (Modeling Programs zip file, .mat, .m files).European Union's Horizon 2020 research and innovation program (grant agreement n° 759282 to Alexander Jones). Gatsby Charitable Foundation grant to Alexander Jones. Leverhulme Trust (Early Career Fellowship to Dr Leah Band, ECF-2012-681). Human Frontier Science Program (Young Investigator Grant, RGY0075/2020 to Leah Band)

Identification of Novel Functionally Important Aromatic Residue Interactions in the Extracellular Domain of the Glycine Receptor.

The extracellular domains (ECDs) of Cys-loop receptors contain many aromatic amino acids, but only relatively few have been well studied. Here we explore the roles of Tyr and Trp residues in the ECD of the glycine receptor and show that four such residues that have not been previously studied (Y24, Y58, W170, and Y197) contribute significantly to the function of the protein. The residues were studied by creating mutant receptors, characterizing them using two-electrode voltage clamp in Xenopus oocytes, and interpreting changes in receptor parameters using structural information about the open and closed states of the receptor. Alanine substitution of all these residues ablates function or increases the glycine EC50. There are also a number of changes in the relative maximal responses to taurine, a partial agonist, compared to glycine. Further mutations, in combination with structural information, suggest Y24 contributes to an anion-π interaction with a binding loop D residue, Y58 to an S-π interaction stabilizing the Cys loop, W170 to hydrophobic interactions stabilizing the hydrophobic interior of the subunit, and Y197 to a hydrogen bond linking binding loops B and C. These interactions appear to be broadly conserved in other Cys-loop receptors. Thus, we have identified new regions of the glycine receptor that are important contributors to receptor activation and are likely also to contribute to function in other members of this important protein family.S.C.R.L. was supported by MRC Grant MR L021676

Recent developments in chemical vapor deposition of 2D magnetic transition metal chalcogenides

In recent years, two-dimensional (2D) magnetic transition metal chalcogenides (TMCs) have attracted tremendous research interests thanks to their intriguing properties that are essential in developing future electronic and spintronic devices in this modernizing era. This review aims to introduce recent developments in the preparation of 2D magnetic TMCs, especially chromium and iron-based chalcogenides, their structures, as well as the related intriguing magnetic phenomena. First, the common crystal structures of magnetic TMCs including both layered and nonlayered structures are introduced. Various chemical vapor deposition strategies for synthesizing 2D magnetic TMCs are then introduced with emphasis on the key synthesis parameters. Moreover, the intriguing physical properties associated with 2D TMCs such as magnetic anisotropy, thickness, and phase-dependent magnetic response as well as stability are summarized. Last but not least, challenges and future research directions are briefly discussed in light of recent advances in the field.Ministry of Education (MOE)National Research Foundation (NRF)Submitted/Accepted versionZ.L. acknowledges support from National Research Foundation Singapore Programme Grants NRF-CRP22-2019-0007, NRF-CRP21-2018-0007, and NRF-CRP22-2019-0004. This research is also supported by the Ministry of Education, Singapore, under its AcRF Tier 3 Programme “Geometrical Quantum Materials” (Grant MOE2018-T3-1-002), and AcRF Tier 1 Grant RG161/19