Biology subjects

Lee, L. K.

Publications and source records attributed to Lee, L. K..

8 recordsLinked to original sources

bioRxiv2026

Contact- and diffusion-based allelopathic interaction of a seaweed with coral holobionts

Shifts from coral-dominated to macroalgal-dominated reef systems have become increasingly common in many coastal regions worldwide. Coral-macroalgal interactions have been shown to be generally detrimental to coral health, with macroalgal allelopathic compounds able to exert serious and even lethal effects on coral at various stages of growth and development. Previous studies have shown that the coral-associated microbial communities play important roles in coral health and mitigating external environmental stress, including macroalgal contact stress. However, it remains unclear whether changes in the coral microbiome have an influence on the Symbiodiniaceae community composition, and if such changes subsequently affect coral health. In this study, we examined changes in both the coral microbiome and the Symbiodiniaceae communities of two Singaporean coral species (Pocillopora acuta and Merulina ampliata) when exposed to both direct and water-mediated macroalgal contact with Lobophora sp. This was investigated using 16S rRNA gene amplicon sequencing to characterize the coral microbiome, and ITS2 variable region sequencing to profile the Symbiodiniaceae communities. Although no significant differences were observed for the coral microbiomes and Symbiodiniaceae communities at both alpha-and beta- diversity levels between control and macroalgal contact treated fragments within each coral species, inter-colony variations in responses to macroalgal contact were observed for both the coral microbiome and Symbiodiniaceae communities of M. ampliata. These results suggest that coral colonies vary in the mechanisms that allow mitigation of the effects of macroalgal contact, and in their resilience to macroalgal-induced stress.

ecology↗

bioRxiv2023

Passivating blunt-ended helices to control monodispersity and multi-subunit assembly of DNA origami structures

DNA origami facilitates the synthesis of bespoke nanoscale structures suitable for a wide range of applications. Effective design requires prevention of uncontrolled aggregation, while still permitting directed assembly of multi-subunit superstructures. Uncontrolled aggregation can be caused by base-stacking interactions between arrays of blunt-ended helices on different structures, which are routinely passivated by incorporating disordered regions as either scaffold loops or poly-nucleotide brushes (usually poly-thymine) at the end of DNA helices. Such disordered regions are ubiquitous in DNA origami structures yet their exact design requirements in different chemical environments are ill defined. In this study, we systematically examine the use of scaffold loops and poly-nucleotide brushes for passivation and for controlling multi-subunit assembly. We assess the dependence of length and sequence for preventing aggregation amidst a titration of MgCl2 concentrations and the suitability of each strategy for enabling controlled multi-subunit assembly. We then introduce a novel strategy where double-stranded DNA helices run orthogonal to arrays of blunt-ended DNA helices forming a steric shield that prevents base stacking. The results define the limitations of each method and important design considerations for achieving monodispersity. For example, poly-thymine brushes are most effective for achieving monodispersity in the broadest conditions whereas scaffold loops can facilitate directed multi-subunit assembly. Finally, orthogonal DNA helices remove the need for disordered regions altogether, prevent aggregation in a broad range of MgCl2 concentrations and facilitate directed multi-subunit assembly. This study expands the design tools available and enables a more informed approach for achieving control of monodispersity and multi-subunit assembly in DNA origami structures.

synthetic biology↗

bioRxiv2023

No isolate, no problem: Using a novel insertion sequence PCR to link rats to human shigellosis cases in an underserved urban community

IntroductionDuring an investigation into a cluster of Shigella flexneri serotype 2a cases in an underserved community, we assessed the relatedness of human and rat S. flexneri isolates utilizing a novel PCR targeting insertion sites (IS-PCR) of mobile elements in the Shigella genome characteristic of the cluster strain. MethodsWhole genome sequences of S. flexneri (n=50) associated with the cluster were analyzed. de novo genome assemblies were analyzed by a Geneious V10.2.6 motif search, and 2 unique IS were identified in all human Shigella sequences of the local cluster. Hydrolysis probe PCR assays were designed to detect these sequences consisting of forward and reverse primers to amplify across each insertion site, and a hydrolysis probe spanning the insertion site. IS-PCR was performed for three Shigella PCR-positive culture-negative rat intestine specimens from this community. ResultsBoth insertion sites were detected in the de novo genome assemblies of all clinical S. flexneri isolates (n=50). Two of the three PCR-positive culture-negative rat samples were positive for both unique IS identified in the human S. flexneri isolates, suggesting that the rat Shigella spp. strains were closely related to the human strains in the cluster. The cycle threshold (Ct) values were >35, indicating that the bacterial load was very low in the rat samples. ConclusionsTwo unique IS were identified in clinical isolates from a community S. flexneri cluster. Both IS targets were identified in PCR-positive (Shigella spp.), culture-negative rat tissue and clinical isolates from humans, indicating relatedness.

microbiology↗

bioRxiv2022

Single cell analysis of gene expression in the substantia nigra pars compacta of a pesticide-induced mouse model of Parkinson's disease

Exposure to pesticides in humans increases the risk of Parkinsons disease (PD), but the mechanisms remain poorly understood. To elucidate these pathways, we dosed C57BL/6J mice with a combination of the pesticides maneb and paraquat (MNPQ). Behavioral analysis revealed motor deficits consistent with PD. Single cell RNA sequencing of substantia nigra pars compacta revealed both cell-type specific genes and genes expressed differentially between pesticide and control, including Fam241b, Emx2os, Bivm, Gm1439, Prdm15 and Rai2. Neurons had the largest number of significant differentially expressed genes, but comparable numbers were found in astrocytes and less so in oligodendrocytes. In addition, network analysis revealed enrichment in functions related to the extracellular matrix. These findings emphasize the importance of support cells in pesticide-induced PD and refocus our attention away from neurons as the sole agent of this disorder.

neuroscience↗

bioRxiv2022

Rapid exchange of stably bound protein and DNA cargo on a DNA origami receptor

Biomolecular complexes can form stable assemblies yet can also rapidly exchange their subunits to adapt to environmental changes. Simultaneously allowing for both stability and rapid exchange expands the functional capacity of biomolecular machines and enables continuous function while navigating a complex molecular world. Inspired by biology, we design and synthesize a DNA origami receptor that exploits multi-valent interactions to form stable complexes that are simultaneously capable of rapid subunit exchange. The system utilizes a mechanism first outlined in the context of the DNA replisome, known as multi-site competitive exchange, and achieves a large separation of time scales between spontaneous subunit dissociation, which requires days, and rapid subunit exchange, which occurs in minutes. In addition, we use the DNA origami receptor to demonstrate stable interactions with rapid exchange of both DNA and protein subunits, thus highlighting the applicability of our approach to arbitrary molecular cargo; an important distinction with canonical toehold exchange between single-stranded DNA. We expect this study to be the first of many that use DNA origami structures to exploit multi-valent interactions for the design and synthesis of a wide range of possible kinetic behaviors.

synthetic biology↗

bioRxiv2021

The clinical utility of two high-throughput 16S rRNA gene sequencing workflows for taxonomic assignment of unidentifiable bacterial pathogens in MALDI-TOF MS

Bacterial pathogens that cannot be identified using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) are occasionally encountered in clinical laboratories. The 16S rRNA gene is often used for sequence-based analysis to identify these bacterial species. Nevertheless, traditional Sanger sequencing is laborious, time-consuming and low-throughput. Here, we compared two commercially available 16S rRNA gene sequencing tests, which are based on Illumina and Nanopore sequencing technologies, respectively, in their ability to identify the species of 172 clinical isolates that failed to be identified by MALDI-TOF MS. Sequencing data were analyzed by respective built-in analysis programs (MiSeq Reporter Software and Epi2me) and BLAST+ (v2.11.0). Their agreement with Sanger sequencing on species-level identification was determined. Discrepancies were resolved by whole-genome sequencing. The diagnostic accuracy of each workflow was determined using the composite sequencing result as the reference standard. Despite the high base-calling accuracy of Illumina sequencing, we demonstrated that the Nanopore workflow had a comparatively higher taxonomic resolution at the species level. Using built-in analysis algorithms, the concordance of Sanger 16S with the Illumina and Nanopore workflows was 33.14% and 87.79%, respectively. The agreement was 65.70% and 83.14%, respectively, when BLAST+ was used for analysis. Compared with the reference standard, the diagnostic accuracy of optimized Nanopore 16S was 96.36%, which was identical to Sanger 16S and was better than Illumina 16S (71.52%). The turnaround time of the Illumina workflow and the Nanopore workflow was 78h and 8.25h, respectively. The per-sample cost of the Illumina and Nanopore workflows was US$28.5 and US$17.7, respectively.

microbiology↗

bioRxiv2019

Design and Synthesis of Pleated DNA Origami Nanotubes with Adjustable Diameters

DNA origami allows for the synthesis of nanoscale structures and machines with nanometre precision and high yields. Tubular DNA origami nanostructures are particularly useful because their geometry facilitates a variety of applications including nanoparticle encapsulation, the construction of artificial membrane pores and as structural scaffolds that can spatially arrange nanoparticles in circular, linear and helical arrays. Here we report a simple computational approach that determines minimally-strained DNA staple crossover locations for arbitrary nanotube internal angles. We apply the method in the design and synthesis of radially symmetric DNA origami nanotubes with arbitrary diameters and DNA helix stoichiometries. These include regular nanotubes where the wall of the structure is composed of a single layer of DNA helices, as well as those with a thicker pleated wall structure that have a greater rigidity and allow for continuously adjustable diameters and distances between parallel helices. We also introduce a DNA origami staple strand routing that incorporates both antiparallel and parallel crossovers and demonstrate its application to further rigidify pleated DNA nanotubes.

synthetic biology↗

bioRxiv2019

Stoichiometric quantification of spatially dense assemblies with qPAINT

Quantitative PAINT (qPAINT) is a useful method for counting well-separated molecules within nanoscale assemblies. But whether cross-reactivity in densely-packed arrangements perturbs measurements is unknown. Here we establish that qPAINT measurements are robust even when target molecules are separated by as little as 3 nm, sufficiently close that single-stranded DNA binding sites can interact.

biophysics↗