bioRxiv Science⌕ Search

Biology subjects

Lay, M.

Publications and source records attributed to Lay, M..

4 recordsLinked to original sources

Real-time single-molecule imaging in zebrafish embryos uncovers non-canonical translation

Precise spatiotemporal control of protein synthesis is essential during embryogenesis, yet directly measuring translation kinetics in vivo remains challenging in vertebrates. In particular, it remains unclear how translation efficiency is determined for key developmental regulators, and which kinetic steps limit their production. Here, we used ALFA array-based nascent chain labeling combined with lattice light-sheet microscopy to visualize bmp2b translation in real time and at single-molecule resolution in early zebrafish embryos. When combined with MS2/MCP labeling to visualize all bmp2b mRNAs, we found that only some of them are being actively translated, suggesting that limited mRNA translation competence contributes to overall translation efficiency. We found that bmp2b translation operates below a maximal initiation regime, as replacement of its UTRs with viral UTRs increases ribosome loading. Furthermore, the bmp2b, but not actb2, 5'UTR supports cap-independent translation with ribosome loading comparable to cap-dependent initiation. Altogether, this approach provides a quantitative in vivo framework to dissect translation kinetics during early vertebrate development.

developmental biology↗

Discovery of neuronal cell types by pairing whole cell reconstructions with RNA expression profiles

Effective classification of neuronal cell types requires both molecular and morphological descriptors to be collected in situ at single-cell resolution. However, current spatial transcriptomics techniques are not compatible with imaging workflows that successfully reconstruct the morphology of complete axonal projections. Here, we introduce a new method, which we call morphoFISH, that combines tissue clearing, submicron whole-brain two photon imaging, and Expansion-Assisted Iterative Fluorescence In Situ Hybridization (EASI-FISH) to assign molecular identities to fully reconstructed neurons in the mouse brain. We used morphoFISH to molecularly identify a previously unknown population of cingulate neurons projecting ipsilaterally to the dorsal striatum and contralaterally to higher-order thalamus. By pairing whole-brain morphometry, improved techniques for nucleic acid preservation and spatial gene expression, morphoFISH offers a quantitative solution for discovery of multimodal cell types and complements existing techniques for characterization of increasingly fine-grained cellular heterogeneity in brain circuits.

neuroscience↗

High fiber, whole foods dietary intervention alters the human gut microbiome but not fecal short-chain fatty acids

Dietary shifts can have a direct impact on the gut microbiome by preferentially selecting for microbes capable of utilizing the various dietary nutrients. Intake of dietary fiber has decreased precipitously in the last century, while consumption of processed foods has increased. Fiber, or microbiota-accessible carbohydrates (MACs), persist in the digestive tract and can be metabolized by specific bacteria encoding fiber degrading enzymes. Digestion of MACs results in the accumulation of short-chain fatty acids (SCFAs) and other metabolic byproducts that are critical to human health. Here, we implemented a two-week dietary fiber intervention aiming for 40-50 grams of fiber per day within the context of a course-based undergraduate research experience (CURE) (n = 20). By coupling shotgun metagenomic sequencing and targeted gas-chromatography mass spectrometry (GC/MS), we found that the dietary intervention significantly altered the composition of individual gut microbiomes, accounting for 8.3% of the longitudinal variability within subjects. Notably, microbial taxa that increased in relative abundance as a result of the diet change included known MAC degraders (i.e., Bifidobacterium and Lactobacillus). We further assessed the genetic diversity within Bifidobacterium, assayed by amplification of the groEL gene. Concomitant with microbial composition changes, we show an increase in the abundance of genes involved in inositol degradation. Despite these changes in gut microbiome composition, we did not detect a consistent shift in SCFA abundance. Collectively, our results demonstrate that on a short-term timescale of two weeks, increased fiber intake can induce compositional changes of the gut microbiome, including an increase in MAC degrading bacteria. IMPORTANCEA profound decrease in the consumption of dietary fiber in many parts of the world in the last century may be associated with the increasing prevalence of Type II diabetes, colon cancer, and other health problems. A typical U.S. diet includes about [~]15 grams of fiber per day, far less fiber than daily recommended allowance. Changes in dietary fiber intake affect human health not only through the uptake of nutrients directly, but also indirectly through changes in the microbial community and their associated metabolism. Here we conducted a two-week diet intervention in healthy young adults to investigate the impact of fiber consumption on the gut microbiome. Participants increased their average fiber consumption by 25 grams/day on average for two weeks. The high fiber diet intervention altered the gut microbiome of the study participants, including increases in known fiber degrading microbes such as Bifidobacterium and Lactobacillus.

microbiology↗

High-Speed Imaging of Paw Withdrawal Reflex to Objectively Assess Pain State in Mice

Rodents are often used for studying chronic pain mechanisms and developing new pain therapeutics, but objectively determining the animals pain state is a major challenge. To improve the precision of using reflexive withdrawal behaviors for interpreting the mouse pain state, we adopted high-speed videography to capture sub-second movement features of mice upon hind paw stimulation. We identified several parameters that are significantly different between behaviors evoked by innocuous and noxious stimuli, and combined them to map the mouse pain state through statistical modeling and machine learning. To test the utility of this approach, we determined the pain state triggered by von Frey hairs (VFHs) and optogenetic activation of two nociceptor populations. Our method reliably assesses the \"pain-like\" probability for each mouse paw withdrawal reflex under all scenarios, highlighting the improved precision of using this high resolution behavior-centered composite methodology to determine the mouse pain state from reflexive withdrawal assays.

neuroscience↗