Biology subjects

Lin, C. H.

Publications and source records attributed to Lin, C. H..

6 recordsLinked to original sources

bioRxiv2025

REverse-transcriptase ACTivity with CRISPR (REACTR) Assay for Ganciclovir Triphosphate Monitoring

BackgroundCytomegalovirus (CMV) can cause severe disease and death in infants and immunocompromised people. Although effective at treating CMV, the first-line drug - ganciclovir (GCV) - has high rates of pharmacokinetic variability which leads to significant rates of underexposure or toxicity. Intracellular concentrations of ganciclovir triphosphate (GCV-TP) - GCVs active anabolite - are associated with dose-dependent neutropenia and could enable dose individualization to improve treatment efficacy and reduce adverse effects. However, GCV-TP is currently measured using liquid chromatography tandem mass spectrometry (LC-MS/MS) which is impractical for routine use, especially in resource-limited settings, because of its high cost, labor-intensiveness, and need for specialized equipment. To address this gap, we adapted the REverse transcriptase ACTivity with CRISPR (REACTR) assay to measure GCV-TP. MethodsWe leveraged our earlier work using REACTR to measure reverse transcriptase (RT) inhibitors used to treat and prevent human immunodeficiency virus (HIV) because GCV-TP serendipitously also inhibits HIV RT. We designed custom DNA templates, primers, and CRISPR complexes to accurately measure GCV-TP spiked into buffer and blood. We evaluated the assays analytical performance with 40 dried blood spots from infants with congenital CMV. ResultsREACTR reproducibly measured clinically relevant GCV-TP concentrations using a simple workflow and equipment that are readily available in many clinical laboratories. REACTR measurements of clinical samples correlated with LC-MS/MS GCV-TP measurements (r = -0.7891; p<0.0001). ConclusionsThis study highlights the potential of REACTR as a rapid and accessible alternative to LC-MS/MS for therapeutic drug monitoring of GCV.

bioengineering↗

bioRxiv2024

Expanding the capillarics toolbox: 3D-printed microfluidic phaseguides and self-coalescence modules

Capillarics are microfluidic circuits that are assembled from individual fluidic elements, powered by surface tension forces encoded by microchannel geometry and surface chemistry, and enable instrument-free pre-programmed automation of multi-step liquid handling processes. 3D printing has recently transformed capillarics by enabling rapid and cost-effective prototyping, provided addition geometric degrees of freedom in multi-level fabrication, and facilitated new design paradigms with greater capabilities than traditional cleanroom fabrication. Despite widespread interest in 3D printing and development of custom high-resolution stereolithography printers for microfluidic applications, fluidic elements that require precise and tunable control over capillary pinning lines -- such as fluidic phaseguides and self-coalescence modules (SCMs) -- have so far only been manufactured with centralized and expensive cleanroom methods. Not only does this limit access to versatile capillaric features to only well-resourced settings, but it also slows innovation and widespread application of these fluid handling technologies. Here we expand the toolbox of 3D-printed capillarics to include phaseguides and SCMs, demonstrating their potential for precise instrument-free control over multi-step liquid handling and reagent rehydration. We employed benchtop stereolithography printers to prototype (up to 50X) scaled-up phaseguides and SCMs and integrated them into a capillaric circuit for inline reagent reconstitution, dynamic fluid control, and sequential drainage. We showcased scalable designs, customizable geometries, and robust self-coalescing flow for larger liquid volumes -- up to 50 L compared with 1.25 L in cleanroom-fabricated SCMs. This work represents a significant advance in democratizing access to microfluidics, with potential for broad applications in diagnostics, assay automation, and organ-on-chip systems.

bioengineering↗

bioRxiv2024

Aging amplifies a gut microbiota immunogenic signature linked to heightened inflammation

Aging is associated with low-grade inflammation that increases the risk of infection and disease, yet the underlying mechanisms remain unclear. Gut microbiota composition shifts with age, harboring microbes with varied immunogenic capacities. We hypothesized the gut microbiota acts as an active driver of low-grade inflammation during aging. Microbiome patterns in aged mice strongly associated with signs of bacterial-induced barrier disruption and immune infiltration, including marked increased levels of circulating lipopolysaccharide (LPS)-binding protein (LBP) and colonic calprotectin. Ex vivo immunogenicity assays revealed that both colonic contents and mucosa of aged mice harbored increased capacity to activate toll-like receptor 4 (TLR4) whereas TLR5 signaling was unchanged. We found patterns of elevated innate inflammatory signaling (colonic Il6, Tnf, Tlr4) and endotoxemia (circulating LBP) in young germ-free mice after 4 weeks of colonization with intestinal contents from aged mice compared with young counterparts, thus providing a direct link between aging-induced shifts in microbiota immunogenicity and host inflammation. Additionally, we discovered that the gut microbiota of aged mice exhibited unique responses to a broad-spectrum antibiotic challenge (Abx), with sustained elevation in Escherichia (Proteobacteria) and altered TLR5 immunogenicity 7 days post-Abx cessation. Together, these data indicate that old age results in a gut microbiota that differentially acts on TLR signaling pathways of the innate immune system. We found that these age-associated microbiota immunogenic signatures are less resilient to challenge and strongly linked to host inflammatory status. Gut microbiota immunogenic signatures should be thus considered as critical factors in mediating chronic inflammatory diseases disproportionally impacting older populations.

immunology↗

bioRxiv2023

Democratizing access to microfluidics: Rapid prototyping of capillary microfluidics with a low-cost masked stereolithography 3D printer

Microfluidics offer user-friendly liquid handling for a range of biochemical applications. 3D printing microfluidics is rapid and cost-effective compared to conventional cleanroom fabrication. Typically, microfluidics are 3D printed using digital light projection (DLP) stereolithography (SLA), but many models in use are expensive ([≥]$10,000 USD), limiting widespread use. Recent liquid crystal display (LCD) technology advancements have provided inexpensive (<$500) SLA 3D printers with sufficient pixel resolution for microfluidic applications. However, there are only a few demonstrations of microfluidic fabrication, limited validation of print fidelity, and no direct comparisons between LCD and DLP printers. We compared a 40 {micro}m pixel resolution DLP printer ([~]$18,000 USD) with a 34.4 {micro}m (<$380) LCD-SLA printer. Consistent with prior work, we observed linear trends between designed and measured channel widths [≥] 4 pixels on both printers, so we calculated accuracy above this size threshold. Using a standard IPA wash resin and optimized parameters for each printer, the average error between designed and measured widths was 2.11 {+/-} 1.26% with the DLP printer and 15.4 {+/-} 2.57% with the 34.4 {micro}m LCD printer. The average coefficient of variation [CV] was [~]2% for both printers. Printing with optimized conditions for a low-cost water wash resin designed for LCD-SLA printers resulted in an average error of 2.53 {+/-} 0.94% with the 34.4 {micro}m LCD printer and 5.35 {+/-} 4.49% with a 22 {micro}m LCD printer. We characterized additional parameters including surface roughness, channel perpendicularity, and light intensity uniformity, and as an application of LCD-printed devices, we demonstrated consistent flow rates in capillaric circuits for self-regulated and self-powered delivery of multiple liquids. In conclusion, LCD printers are an inexpensive alternative for fabricating microfluidics, with minimal differences in fidelity and accuracy compared with a 20X more expensive DLP printer.

scientific communication and education↗

bioRxiv2023

How the dominant reading direction changes parafoveal processing: A combined EEG/eye-tracking study

Reading directions vary across writing systems. Through long-term experience readers adjust their visual systems to the dominant reading direction in their writing systems. However, little is known about the neural correlates underlying these adjustments because different writing systems do not just differ in reading direction, but also regarding visual and linguistic properties. Here, we took advantage that Chinese is read to different degrees in left-right or top-down directions in different regions. We investigated visual word processing in participants from Taiwan (both top-down and left-right directions) and from mainland China (only left-right direction). Combined EEG/eye tracking was used together with a saccade-contingent parafoveal preview manipulation to investigate neural correlates, while participants read 5-word lists. Fixation-related potentials (FRPs) showed a reduced late N1 effect (preview positivity), but this effect was modulated by the prior experience with a specific reading direction. Results replicate previous findings that valid previews facilitate visual word processing, as indicated by reduced FRP activation. Critically, the results indicate that this facilitation effect depends on experience with a given reading direction, suggesting a specific mechanism how cultural experience shapes the way people process visual information.

neuroscience↗

bioRxiv2021

Trisomy 21 induces pericentrosomal crowding disrupting early stages of primary ciliogenesis and mouse cerebellar development

Primary cilia are signaling organelles essential for development and homeostasis. Loss of primary cilia is lethal, and decreased or defective cilia cause multisystemic conditions called ciliopathies. Down syndrome shares clinical overlap with ciliopathies. We previously showed that trisomy 21 diminishes primary cilia formation and function due to elevated Pericentrin, a centrosome protein encoded on chromosome 21. Pericentrin is mislocalized, creating aggregates that disrupt pericentrosomal trafficking and microtubule organization. Here, we examine the cilia-related molecules and pathways disrupted in trisomy 21 and their in vivo phenotypic relevance. Utilizing ciliogenesis time course experiments, we reveal how Pericentrin, microtubule networks, and components of ciliary vesicles are reorganized for ciliogenesis in euploid cells. Early in ciliogenesis, chromosome 21 polyploidy results in elevated Pericentrin and microtubule networks away from the centrosome that ensnare MyosinVA and EHD1, blocking mother centriole uncapping that is essential for ciliogenesis. Ciliated trisomy 21 cells have persistent trafficking defects that reduce transition zone protein localization, which is critical for Sonic hedgehog signaling. Sonic hedgehog signaling is decreased and anticorrelates with Pericentrin levels in trisomy 21 primary mouse embryonic fibroblasts. Finally, we observe decreased ciliation in vivo. A mouse model of Down syndrome with elevated Pericentrin has fewer primary cilia in cerebellar granule neuron progenitors and thinner external granular layers. Our work reveals that elevated Pericentrin in trisomy 21 disrupts multiple early steps of ciliogenesis and creates persistent trafficking defects in ciliated cells. This pericentrosomal crowding results in signaling defects consistent with the neurological deficits found in individuals with Down syndrome.

cell biology↗