Biology subjects

Lam, A. W.

Publications and source records attributed to Lam, A. W..

6 recordsLinked to original sources

bioRxiv2025

A multiplex extracellular interactome screening method employing high-avidity nanoparticles

Metazoan cells signal to each other via direct contact between cell surface proteins (CSPs) and by interactions of CSP receptors with secreted ligands. CSP extracellular domain (ECD) interactions control organ development and physiology and are perturbed in disease states. However, because they cannot be accurately assessed using standard high-throughput screening techniques, they are underrepresented in protein interaction databases. Many ECD interactions are of low affinity, and their detection in vitro requires taking advantage of avidity effects, typically by multimerization of fusion proteins. Assays that test only one or a few interactions in each binding reaction are inadequate for global interactome screening. Here we describe a new multiplex method that uses purified dimeric ECD fusion proteins coupled to 60-mer nanoparticles as soluble prey, and the same dimers coupled to spectrally distinguishable fluorescent microspheres (beads) as bait. We add one prey to a mixture of up to 500 baits in a single well, then use a Luminex FLEXMAP 3D (FM3D) instrument to read out bait identity and prey binding. The FM3D measures the fluorescent dye ratio for each bead and simultaneously determines the amount of epitope-tagged prey bound to that bead. We use the method, denoted as the Multiplex Interactome Assay (MPIA), to analyze a proof-of-concept (PoC) set of 41 CSPs and secreted protens that is derived from larger collections examined in two interactome screens that used ELISA-based binding assays. By analyzing interactions among PoC proteins, we compared the MPIA with earlier screening methods. The MPIA has a dynamic range that is at least 30-fold greater than ELISA-based assays and appears to be more sensitive. By coupling the MPIA to an automated protein production and purification platform, we hope to be able to conduct a screen for interactions among thousands of human CSPs and secreted ligands.

molecular biology↗

bioRxiv2025

An Automated End-to-End Workflow for Production of Secreted Proteins in Transfected Mammalian Cells

The advancement of automation technologies has helped to enable a surge in large-scale screening efforts across fields such as molecular biology, protein biochemistry, cell biology, and structural biology. In the context of this "omics"-driven research, there is a need to generate automation platforms that are more flexible and less expensive, so that they can be utilized for basic research conducted by small groups. A key challenge in automation lies in developing methods that can replicate fine motor techniques that are normally performed manually by researchers at the bench. We are engaged in a large-scale project to map interactions among human cell-surface and secreted proteins and assess their effects on cells. This project involves production of a library of more than 2000 recombinant His-tagged fusion proteins secreted from transfected Expi293 cells. To execute such a project with a small group at an academic institution required construction of an affordable automated system that could also be used by other investigators. This led us to develop a high-throughput, 96-well format automation platform for end-to-end protein production. The workflow includes transformation of E. coli, plasmid DNA preparation, transient transfection, protein purification, desalting and buffer exchange, protein quantification, and normalization of protein concentrations, resulting in assay-ready proteins. The system is built around an in-house engineered modular robotic platform that integrates liquid handling with a suite of interchangeable plug-and-play mobile enclosed device modules. Housed within a BSL-2 sterile environment, the platform enables flexible, fully automated workflows and can be readily customized for diverse user-defined protocols.

biochemistry↗

bioRxiv2023

Primate-conserved Carbonic Anhydrase IV and murine-restricted Ly6c1 are new targets for crossing the blood-brain barrier

The blood-brain barrier (BBB) presents a major challenge to delivering large molecules to study and treat the central nervous system (CNS). This is due in part to the scarcity of effective targets for BBB crossing, the identification of which is the crucial first step of drug development. Here, we leveraged a panel of adeno-associated viruses (AAVs) previously identified through directed evolution for improved BBB transport to reverse engineer protein targets for enhanced BBB crossing. We identify both murine-restricted Ly6c1 and primate-conserved carbonic anhydrase IV (Car4; CA4) as novel receptors for crossing the BBB. We demonstrate how these receptors can unlock new experimental and computational target-focused engineering strategies by creating the enhanced Ly6c1-binding vector AAV-PHP.eC and by applying AlphaFold2-enabled in silico methods to rank capsids against identified receptors and generate capsid-receptor binding models. Here, with Car4, we add a completely new receptor to the very short list currently available for crossing the BBB in humans and, with Ly6c1, we validate a pipeline for receptor-targeted engineering. The identification of Car4/CA4 and structural insights from computational modeling provide new paths toward human brain-penetrant chemicals (drugs) and biologicals (including gene delivery).

bioengineering↗

bioRxiv2021

Chromosomal-level genome assembly of the bioluminescent cardinalfishSiphamia tubifer, an emerging model for symbiosis research

The bioluminescent symbiosis between the sea urchin cardinalfish Siphamia tubifer (Kurtiformes: Apogonidae) and the luminous bacterium Photobacterium mandapamensis is an emerging vertebrate-bacteria model for the study of microbial symbiosis. However, there is little genetic data available for the host fish, limiting the scope of potential research that can be carried out with this association. In this study, we present a chromosomal-level genome assembly of S. tubifer using a combination of PacBio HiFi sequencing and Hi-C technologies. The final genome assembly was 1.2 Gb distributed on 23 chromosomes and contained 32,365 protein coding genes with a BUSCO completeness score of 99%. A comparison of the S. tubifer genome to that of another non-luminous cardinalfish revealed a high degree of synteny, whereas a similar comparison to a more distant relative in the Gobiiformes order revealed a fusion of two chromosomes in the cardinalfish genomes. An additional comparison of orthologous clusters among these three genomes revealed a set of 710 clusters that were unique to S. tubifer in which 23 GO pathways were significantly enriched, including several relating to host-microbe interactions and one involved in visceral muscle development, which could be related to the musculature involved in the gut-associated light organ of S. tubifer. We also assembled the complete mitogenome of S. tubifer and discovered both an inversion in the WANCY tRNA gene region resulting in a WACNY gene order as well as heteroplasmy in the length of the control region for this individual. A phylogenetic analysis based on the whole mitochondrial genome indicated that S. tubifer is divergent from the rest of the cardinalfish family, bringing up questions of the involvement of the bioluminescent symbiosis in the initial divergence of the ancestral Siphamia species. This draft genome assembly of S. tubifer will enable future studies investigating the evolution of bioluminescence in fishes as well as candidate genes involved in the symbiosis and will provide novel opportunities to use this system as a vertebrate-bacteria model for symbiosis research.

genomics↗

bioRxiv2021

How the Easter Egg Weevils Got Their Spots: Phylogenomics reveals Müllerian Mimicry in Pachyrhynchus (Coleoptera, Curculionidae).

The evolutionary origins of mimicry in the Easter Egg weevil, Pachyrhynchus, have fascinated researchers since first noted more than a century ago by Alfred Russel Wallace. Mullerian mimicry, or mimicry in which two or more distasteful species look similar, is widespread throughout the animal kingdom. Given the varied but discrete color patterns in Pachyrhynchus, this genus presents one of the best opportunities to study the evolution of both perfect and imperfect mimicry. We analyzed more than 10,000 UCE loci using a novel partitioning strategy to resolve the relationships of closely related species in the genus. Our results indicate that many of the mimetic color patterns observed in sympatric species are due to convergent evolution. We suggest that this convergence is driven by frequency-dependent selection.

evolutionary biology↗

bioRxiv2020

The Easter Egg Weevil (Pachyrhynchus) genome reveals synteny in Coleoptera across 200 million years of evolution

Patterns of genomic architecture across insects remain largely undocumented or decoupled from a broader phylogenetic context. For instance, it is unknown whether translocation rates differ between insect orders? We address broad scale patterns of genome architecture across Insecta by examining synteny in a phylogenetic framework from open source insect genomes. To accomplish this, we add a chromosome level genome to a crucial lineage, Coleoptera. Our assembly of the Pachyrhynchus sulphureomaculatus genome is the first chromosome scale genome for the hyperdiverse Phytophaga lineage and currently the largest insect genome assembled to this scale. The genome is significantly larger than those of other weevils, and this increase in size is caused by repetitive elements. Our results also indicate that, among beetles, there are instances of long-lasting (>200 Ma) localization of genes to a particular chromosome with few translocation events. While some chromosomes have a paucity of translocations, intra-chromosomal synteny was almost absent, with gene order thoroughly shuffled along a chromosome. To place our findings in an evolutionary context, we compared syntenic patterns across Insecta. We find that synteny largely scales with clade age, with younger clades, such as Lepidoptera, having especially high synteny. However, we do find subtle differences in the maintenance of synteny and its rate of decay among the insect orders.

genomics↗