Biology subjects

Wong, S. S.

Publications and source records attributed to Wong, S. S..

5 recordsLinked to original sources

bioRxiv2024

High-throughput sequencing-based neutralization assay reveals how repeated vaccinations impact titers to recent human H1N1 influenza strains

The high genetic diversity of influenza viruses means that traditional serological assays have too low throughput to measure serum antibody neutralization titers against all relevant strains. To overcome this challenge, we have developed a sequencing-based neutralization assay that simultaneously measures titers against many viral strains using small serum volumes via a workflow similar to traditional neutralization assays. The key innovation is to incorporate unique nucleotide barcodes into the hemagglutinin (HA) genomic segment, and then pool viruses with numerous different barcoded HA variants and quantify infectivity of all of them simultaneously using next-generation sequencing. With this approach, a single researcher performed the equivalent of 2,880 traditional neutralization assays (80 serum samples against 36 viral strains) in approximately one month. We applied the sequencing-based assay to quantify the impact of influenza vaccination on neutralization titers against recent human H1N1 strains for individuals who had or had not also received a vaccine in the previous year. We found that the viral strain specificities of the neutralizing antibodies elicited by vaccination vary among individuals, and that vaccination induced a smaller increase in titers for individuals who had also received a vaccine the previous year--although the titers six months after vaccination were similar in individuals with and without the previous-year vaccination. We also identified a subset of individuals with low titers to a subclade of recent H1N1 even after vaccination. This study demonstrates the utility of high-throughput sequencing-based neutralization assays that enable titers to be simultaneously measured against many different viral strains. We provide a detailed experimental protocol (DOI: https://dx.doi.org/10.17504/protocols.io.kqdg3xdmpg25/v1) and a computational pipeline (https://github.com/jbloomlab/seqneut-pipeline) for the sequencing-based neutralization assays to facilitate the use of this method by others.

microbiology↗

bioRxiv2024

Charting Single Cell Lineage Dynamics and Mutation Networks via Homing CRISPR

Single cell lineage tracing, essential for unraveling cellular dynamics in disease evolution is critical for developing targeted therapies. CRISPR-Cas9, known for inducing permanent and cumulative mutations, is a cornerstone in lineage tracing. The novel homing guide RNA (hgRNA) technology enhances this by enabling dynamic retargeting and facilitating ongoing genetic modifications. Charting these mutations, especially through successive hgRNA edits, poses a significant challenge. Our solution, LINEMAP, is a computational framework designed to trace and map these mutations with precision. LINEMAP meticulously discerns mutation alleles at single-cell resolution and maps their complex interrelationships through a mutation evolution network. By utilizing a Markov Process model, we can predict mutation transition probabilities, revealing potential mutational routes and pathways. Our reconstruction algorithm, anchored in the Markov models attributes, reconstructs cellular lineage pathways, shedding light on the cells evolutionary journey to the minutiae of single-cell division. Our findings reveal an intricate network of mutation evolution paired with a predictive Markov model, advancing our capability to reconstruct single-cell lineage via hgRNA. This has substantial implications for advancing our understanding of biological mechanisms and propelling medical research forward.

bioinformatics↗

bioRxiv2021

Centriole distal-end proteins CP110 and Cep97 influence cartwheel growth at the proximal-end of centrioles

Centrioles are composed of a central cartwheel tethered to nine-fold symmetric microtubule (MT) blades. The centriole cartwheel and MTs are thought to grow from opposite ends of these organelles, so it is unclear how they coordinate their assembly. We previously showed that an oscillation of Polo-like kinase 4 (Plk4) helps to initiate and time the growth of the cartwheel at the proximal end. Here, we show that CP110 and Cep97 form a complex close to the distal-end of the centriole MTs whose levels rise and fall as the new centriole MTs grow, entrained by the core Cdk/Cyclin oscillator that drives the nuclear divisions in these embryos. These CP110/Cep97 dynamics, however, do not appear to time the period of centriole MT growth directly. Instead, we find that changing the levels of CP110/Cep97 alters the Plk4 oscillation and the growth of the cartwheel at the proximal end. These findings reveal an unexpected crosstalk between factors normally concentrated at opposite ends of the growing centrioles, which may help to coordinate centriole growth.

cell biology↗

bioRxiv2020

A Rapid COVID-19 RT-PCR Detection Assay for Low Resource Settings

Quantitative reverse transcription polymerase chain reaction (RT-qPCR) assay is the gold standard recommended to test for acute SARS-CoV-2 infection. It has been used by the Centers for Disease Control and Prevention (CDC) and several other companies in their Emergency Use Authorization (EUA) assays. RT-qPCR requires expensive equipment such as RNA isolation instruments and real-time PCR thermal cyclers, which are not available in many low resource settings and developing countries. As a pandemic, COVID-19 has quickly spread to the rest of the world. Many underdeveloped and developing counties do not have the means for fast and accurate COVID-19 detection to control this outbreak. Using COVID-19 positive clinical specimens, we demonstrated that RT-PCR assays can be performed in as little as 12 minutes using untreated samples, heat-inactivated samples, or extracted RNA templates. Rapid RT-PCR was achieved using thin-walled PCR tubes and a setup including sous vide immersion heaters/circulators. Our data suggest that rapid RT-PCR can be implemented for sensitive and specific molecular diagnosis of COVID-19 in situations where sophisticated laboratory instruments are not available.

molecular biology↗

bioRxiv2020

The Potential Use of Unprocessed Sample for RT-qPCR Detection of COVID-19 without an RNA Extraction Step

Quantitative reverse transcription polymerase chain reaction (RT-qPCR) assay is the gold standard recommended to test for acute SARS-CoV-2 infection.1-4 It has been used by the Centers for Disease Control and Prevention (CDC) and several other companies in their Emergency Use Authorization (EUA) assays. With many PCR-based molecular assays, an extraction step is routinely used as part of the protocol. This step can take up a significant amount of time and labor, especially if the extraction is performed manually. Long assay time, partly caused by slow sample preparation steps, has created a large backlog when testing patient samples suspected of COVID-19. Using flu and RSV clinical specimens, we have collected evidence that the RT-qPCR assay can be performed directly on patient sample material from a nasal swab immersed in virus transport medium (VTM) without an RNA extraction step. We have also used this approach to test for the direct detection of SARS-CoV-2 reference materials spiked in VTM. Our data, while preliminary, suggest that using a few microliters of these untreated samples still can lead to sensitive test results. If RNA extraction steps can be omitted without significantly affecting clinical sensitivity, the turn-around time of COVID-19 tests and the backlog we currently experience can be reduced drastically. Next, we will confirm our findings using patient samples.

molecular biology↗