Chang, C.-M. | Search bioRxiv

bioRxiv2022

PKD1 and PKD2 mRNA cis-inhibition drives polycystic kidney disease progression

Autosomal dominant polycystic kidney disease (ADPKD), among the most common human genetic conditions and a frequent etiology of kidney failure, is primarily caused by heterozygous PKD1 mutations. Kidney cyst formation ensues when the PKD1 dosage falls below a critical threshold. However, no framework exists to harness the remaining allele or reverse PKD1 decline. Here, we show that mRNAs produced by the noninactivated PKD1 allele are cis-repressed via their 3-UTR miR-17 binding element. Eliminating this motif (Pkd1{Delta}17) improves mRNA stability, raises Polycystin-1 levels, and alleviates cyst growth in cellular, ex vivo, and mouse PKD models. Remarkably, Pkd2 is also autoinhibited via its 3-UTR miR-17 motif, and Pkd2{Delta}17-induced Polycystin-2 derepression partly compensates and retards cyst growth in Pkd1-mutant models. Moreover, acutely blocking Pkd1/2 cis-inhibition, including after cyst onset, attenuates murine PKD. Finally, PKD1{Delta}17 or PKD2{Delta}17 alleles revert cyst-pathogenic sequala in patient-derived primary ADPKD cultures. Thus, evading 3-UTR cis-interference and enhancing PKD1/2 mRNA translation is a potentially mutation-agnostic ADPKD-arresting approach.

genetics↗

bioRxiv2020

Determination of Bacterial Surface Charge Density Via Saturation of Adsorbed Ions

Bacterial surface charge is a critical characteristic of the cells interfacial physiology that influences how the cell interacts with the local environment. A direct, sensitive, and accurate experimental technique capable of quantifying bacterial surface charge is needed to better understand molecular adaptations in interfacial physiology in response to environmental changes. We introduce here the method of second harmonic light scattering (SHS) which is capable of detecting the number of molecular ions adsorbed as counter charges on the exterior bacterial surface, thereby providing a measure of the surface charge. In this first demonstration, we detect the small molecular cation, malachite green, electrostatically adsorbed on the surface of representative strains of Gram-positive and Gram-negative bacteria. Surprisingly, the SHS deduced molecular transport rates through the different cellular ultra-structures are revealed to be nearly identical. However, the adsorption saturation densities on the exterior surfaces of the two bacteria were shown to be characteristically distinct. The negative charge density of the lipopolysaccharide coated outer surface of Gram-negative E. coli (8.7{+/-}1.7 nm-2) was deduced to be seven times larger than that of the protein surface layer of Gram-positive L. rhamnosus (1.2{+/-}0.2 nm-2). The feasibility of SHS deduced bacterial surface charge density for Gram-type differentiation is presented. STATEMENT of SIGNIFICANCEBacterial surface charge density is an important physiological characteristic which determines how the cell interacts with its local environment. Directly measuring the surface charge density, however, is experimentally non-trivial. In this work, we report an experimental method, second harmonic light scattering, that can directly and accurately quantify the surface charge density of individual living bacteria. This is achieved by measuring the number of molecular ions electrostatically adsorbed on the exterior cellular surface as counter charges. It is found that the negative charge density of a representative Gram-negative bacterium is 7 times larger than a representative Gram-positive bacterium. It is suggested that this disparity of surface charge density can be exploited as a basis for Gram-classification of bacteria.

biophysics↗