Biology subjects

Lee, V. S.

Publications and source records attributed to Lee, V. S..

4 recordsLinked to original sources

bioRxiv2026

What Can We Count On? Performance of Microplate Cell Counting Assays in 2D Monolayer and 3D ECM-based In Vitro Tumour Models

Accurate cell counting is essential in tissue engineering and cancer research. The ongoing transition towards advanced 3D in vitro tumour models raises a question about the validity of the standard cell counting protocols, particularly in the systems containing extracellular matrix-based scaffolds. Here, we provide a quantitative analysis of the performance of three popular plate reader-based cell counting/viability assays, such as the Alamar Blue, MTT, CellTiter Glo 3D assays, in 2D monolayer and 3D scaffold-based cultures of U251 human glioblastoma cells, including cell-laden Matrigel plugs, and original tissue engineering constructs based on the decellularised sheep brain scaffolds. We quantitatively characterized the assays linearity, precision, biological and technical reproducibility, proportionality, and inter-assay agreement. The study revealed that assays performance is highly platform-dependent, with 2D cultures allowing significantly more precise and reliable measurements than in 3D ECM scaffold-based cultures. The numerical results provided in this study can help researchers make informed decisions when working with 3D scaffold-based in vitro tumour models and for other tissue engineering purposes where precise cell counting is essential. ToC O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=184 SRC="FIGDIR/small/720021v1_ufig1.gif" ALT="Figure 1"> View larger version (39K): org.highwire.dtl.DTLVardef@16018d9org.highwire.dtl.DTLVardef@1ff7d6dorg.highwire.dtl.DTLVardef@838021org.highwire.dtl.DTLVardef@1510d5b_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗

bioRxiv2023

Cingulate cortex facilitates auditory perception under challenging listening conditions

We often exert greater cognitive resources (i.e., listening effort) to understand speech under challenging acoustic conditions. This mechanism can be overwhelmed in those with hearing loss, resulting in cognitive fatigue in adults, and potentially impeding language acquisition in children. However, the neural mechanisms that support listening effort are uncertain. Evidence from human studies suggest that the cingulate cortex is engaged under difficult listening conditions, and may exert top-down modulation of the auditory cortex (AC). Here, we asked whether the gerbil cingulate cortex (Cg) sends anatomical projections to the AC that facilitate perceptual performance. To model challenging listening conditions, we used a sound discrimination task in which stimulus parameters were presented in either Easy or Hard blocks (i.e., long or short stimulus duration, respectively). Gerbils achieved statistically identical psychometric performance in Easy and Hard blocks. Anatomical tracing experiments revealed a strong, descending projection from layer 2/3 of the Cg1 subregion of the cingulate cortex to superficial and deep layers of primary and dorsal AC. To determine whether Cg improves task performance under challenging conditions, we bilaterally infused muscimol to inactivate Cg1, and found that psychometric thresholds were degraded for only Hard blocks. To test whether the Cg-to-AC projection facilitates task performance, we chemogenetically inactivated these inputs and found that performance was only degraded during Hard blocks. Taken together, the results reveal a descending cortical pathway that facilitates perceptual performance during challenging listening conditions. Significance StatementSensory perception often occurs under challenging conditions, such a noisy background or dim environment, yet stimulus sensitivity can remain unaffected. One hypothesis is that cognitive resources are recruited to the task, thereby facilitating perceptual performance. Here, we identify a top-down cortical circuit, from cingulate to auditory cortex in the gerbils, that supports auditory perceptual performance under challenging listening conditions. This pathway is a plausible circuit that supports effortful listening, and may be degraded by hearing loss.

neuroscience↗

bioRxiv2019

Electrostatic interactions at the five-fold axis alter heparin-binding phenotype and drive EV-A71 virulence in mice

Enterovirus A71 (EV-A71) causes hand, foot and mouth disease epidemics with neurological complications and fatalities. However, the neuropathogenesis of EV-A71 remains poorly understood. In mice, adaptation and virulence determinants have been mapped to mutations at VP1-145, VP1-244 and VP2-149. We hypothesized that heparin-binding phenotype shapes EV-A71 virulence in mice. We constructed six viruses with varying residues at VP1-98, VP1-145 (which are both heparin-binding determinants) and VP2-149 (based on the wild type 98E/145Q/149K, termed EQK) to generate KQK, KEK, EEK, EEI and EQI variants. We demonstrated that the weak heparin-binder EEI was highly lethal in mice. The initially strong heparin-binding EQI variant acquired an additional mutation VP1-K244E, which confers weak heparin-binding phenotype resulting in elevated viremia and increased brain inflammation and virus antigens in mice, with subsequent high virulence. EEI and EQI-K244E variants inoculated into mice disseminated efficiently and displayed high viremia. Increasing polymerase fidelity and impairing recombination of EQI attenuated virulence, suggesting the importance of population diversity in EV-A71 pathogenesis in vivo. Combining in silico docking and deep sequencing approaches, we inferred that virus population diversity is shaped by electrostatic interactions at the five-fold axis of the virus surface. Electrostatic surface charges facilitate virus adaptation by generating poor heparin-binding variants for better in vivo dissemination in mice, likely due to reduced adsorption to heparin-rich peripheral tissues, which ultimately results in increased neurovirulence. The dynamic switching from heparin-binding to weak heparin-binding phenotype in vivo explained the neurovirulence of EV-A71.\n\nAuthor summaryEnterovirus A71 (EV-A71) is the primary cause of hand, foot and mouth disease, and it can also infect the central nervous system and cause fatal outbreaks in young children. EV-A71 pathogenesis remains elusive. In this study, we demonstrated that EV-A71 variants with strong affinity to heparan sulfate (heparin) have a growth advantage in tissue culture, but are disadvantageous in vivo. When inoculated into mice, strong heparin-binding virus variants are more likely to be adsorbed to peripheral tissues, resulting in impaired ability to disseminate and being cleared from the bloodstream rapidly. The lower viremia level resulted in no neuroinvasion. In contrast, weak heparin-binding variants show greater levels of viremia, dissemination and subsequent neurovirulence in mice. We also provide evidence that the ability of EV-A71 to bind heparin is mediated by electrostatic surface charges due to amino acids on the virus capsid surface. In mice, EV-A71 undergoes adaptive mutation to acquire greater negative surface charges, thus generating new virulent variants with weak heparin-binding which allows greater viral spread. Our study underlines the importance of electrostatic surface charges in shaping EV-A71 virulence.

microbiology↗

bioRxiv2018

A Cross-Platform Comparison of Student’s Perceptions of the Learning Environment in an Introductory Microbiology Course

AbstractStudents positive perceptions of the learning environment increase retention and persistence in STEM disciplines. This study presents results from a Learning Environment Questionnaire administered at the beginning and end of the semester in a redesigned general/introductory microbiology course offered in traditional lecture, flipped and online sections. Split-sample t-tests and chi-squared tests were used for cross-section and within section analyses, respectively. The findings support the study hypothesis that student perceptions of the learning environment will vary as a function of platform. This work demonstrates the additional effect of the time in the semester when students complete the questionnaire and this effect on students perceptions over the course of the semester relative to their initial perceptions. The results of this study offer insight on student perceptions of the learning environment as universities embrace online education in introductory and gateway courses as a response to rising student enrollments and diminishing resources.

scientific communication and education↗