Biology subjects

Mes, W.

Publications and source records attributed to Mes, W..

2 recordsLinked to original sources

bioRxiv2026

Zebrafish larval nitrogen excretion is flexible and resilient to loss of rhesus glycoproteins

Nitrogenous waste excretion is essential for all developmental stages of fish. Embryonic fish excrete urea, transitioning to cutaneous and later branchial ammonia excretion. In zebrafish, ammonia excretion involves rhesus glycoproteins Rhbg and Rhcgb in keratinocytes and ionocytes, but the developmental moment they appear in the gill remains unclear. Potential redundancy between Rhbg and Rhcgb in ammonia excretion is also not fully investigated, nor is the difference in response to low pH. We hypothesized that rhesus glycoproteins are partially redundant, and that they differ in their response to low pH as ammonia excretion enables ionocytes to exchange Na+ and H+ (Rh-NHE-metabolon). We predicted that a loss of rhbg or rhcgb induces compensatory responses. We characterized the transition from urea to branchial ammonia excretion from 0 to 8 days-post fertilization (dpf) and the response to pH 5.0 on the expression and localization of rhesus glycoproteins in control zebrafish and rhbg or rhcgb-crispants. Effects of high external ammonia (HEA, 500 M NH4Cl) and 10 mM HEPES-buffering were further characterized in rhcgb-crispants. Rhag and Rhbg appeared in the gill at 5 dpf, while Rhcgb appeared at 6 dpf. A loss of rhbg or rhcgb did not impact baseline N-excretion, illustrating that zebrafish can maintain ammonia excretion without the full complement of rhesus glycoproteins. We observed no compensatory increase in rhesus glycoproteins, but expression of the transporter hippocampus-abundant transcript 1b increased. HEA-exposed rhcgb-crispants switched to urea as primary nitrogen waste. Together, these findings underline the plasticity of the larval in dealing with nitrogenous waste.

physiology↗

bioRxiv2020

Optimized DNA isolation method for microbiome analysis of human tissues

Recent advances in microbiome sequencing have rendered new insights into the role of the microbiome in human health with potential clinical implications. Unfortunately, developments in the field of tissue microbiomes have been hampered by the presence of host DNA in isolates which interferes with the analysis of the bacterial content. Here, we present a DNA isolation protocol from tissue samples including reduction of host DNA without distortion of microbial abundance profiles. We evaluated which concentrations of Triton and saponin lyse host cells and leave bacterial cells intact, which was combined with DNAse treatment to deplete released host DNA. We applied our protocol to extract microbial DNA from ex vivo and in vivo acquired human colon biopsies ([~]2-5 mm in size) and assessed the relative abundance of bacterial and human DNA by qPCR. Saponin at a concentration of 0.0125% in PBS lysed host cells, resulting in a 4.5-fold enrichment of bacterial DNA while preserving the relative abundance of Firmicutes, Bacteroidetes, {gamma}-Proteobacteria and Actinobacteria. Our protocol combined with shotgun metagenomic sequencing revealed a colon tissue microbiome profile with a Shannon diversity index of 3.2 and an UniFrac distance of 0.54, which is comparable to reported numbers based on amplicon sequencing. Hereby, we present the first protocol for enriching bacterial DNA from tissue biopsies that allows efficient isolation of rigid Gram-positive bacteria without depleting the more sensitive Gram-negative bacteria. Our protocol facilitates analysis of a wide spectrum of bacteria of clinical tissue samples improving their applicability for microbiome research.

microbiology↗