Liou, W. | bioRxiv Science

bioRxiv2025

EVOH Hollow Fiber Encapsulation of Individual Caenorhabditis elegans for Transmission Electron Microscopy

We present a streamlined protocol for encapsulating individual Caenorhabditis elegans specimens for thin-section transmission electron microscopy (TEM) using ethylene vinyl alcohol (EVOH) copolymer hollow fibers. These transparent fibers, featuring a 175 m inner diameter, 25 m wall thickness, and an approximate molecular weight cutoff of 30 kDa, possess both hydrophilic and hydrophobic properties. This amphiphilic nature helps maintain a potent lumen structure even in dry, open air, enabling precise handling and orientation of small specimens prior to resin polymerization and eliminating the need for remounting. The protocol includes specimen transfer using wire loops, microwave-assisted chemical pre-fixation, dissection on a soft silicone substrate with micro-scissors, and manipulation of hollow fibers using insect pins. This approach preserves the identity of individual specimens throughout processing. During ultramicrotomy, the fiber serves as a visual guide, enhancing alignment and reproducibility of sectioning. As proof of principle, we demonstrate well-preserved ultrastructural features in chemically fixed, microwave- assisted resin-embedded C. elegans specimens. This method offers a robust and reproducible alternative to conventional gelatinous media encapsulation, particularly suited for TEM analysis of scarce or small biological samples.

cell biology↗

bioRxiv2024

Long-term ketogenic diet causes hyperlipidemia, liver dysfunction, and glucose intolerance from impaired insulin trafficking and secretion in mice

A ketogenic diet (KD) is a very low-carbohydrate, very high-fat diet proposed to treat obesity and type 2 diabetes. While KD grows in popularity, its effects on metabolic health are understudied. Here we show that, in male and female mice, while KD protects against weight gain and induces weight loss, over long-term, mice develop hyperlipidemia, hepatic steatosis, and severe glucose intolerance. Unlike high fat diet-fed mice, KD mice are not insulin resistant and have low levels of insulin. Hyperglycemic clamp and ex vivo GSIS revealed cell-autonomous and whole-body impairments in insulin secretion. Major ER/Golgi stress and disrupted ER-Golgi protein trafficking was indicated by transcriptomic profiling of KD islets and confirmed by electron micrographs showing a dilated Golgi network likely responsible for impaired insulin granule trafficking and secretion. Overall, our results suggest long-term KD leads to multiple aberrations of metabolic parameters that caution its systematic use as a health promoting dietary intervention.

physiology↗