Glow-in-the-Dark Infectious Disease Diagnostics Using CRISPR-Cas9-Based Split Luciferase Complementation.

Nucleic acid detection methods based on CRISPR and isothermal amplification techniques show great potential for point-of-care diagnostic applications. However, most current methods rely on fluorescent or lateral flow assay readout, requiring external excitation or postamplification reaction transfer. Here, we developed a bioluminescent nucleic acid sensor (LUNAS) platform in which target dsDNA is sequence-specifically detected by a pair of dCas9-based probes mediating split NanoLuc luciferase complementation. LUNAS is easily integrated with recombinase polymerase amplification (RPA), providing attomolar sensitivity in a rapid one-pot assay. A calibrator luciferase is included for a robust ratiometric readout, enabling real-time monitoring of the RPA reaction using a simple digital camera. We designed an RT-RPA-LUNAS assay that allows SARS-CoV-2 RNA detection without the need for cumbersome RNA isolation and demonstrated its diagnostic performance for COVID-19 patient nasopharyngeal swab samples. Detection of SARS-CoV-2 from samples with viral RNA loads of ∼200 cp/µL was achieved within ∼20 min, showing that RPA-LUNAS is attractive for point-of-care infectious disease testing.

Comprehensive analytical and clinical evaluation of a RNA extraction-free saliva-based molecular assay for SARS-CoV-2.

Standard SARS-CoV-2 testing protocols using nasopharyngeal/throat (NP/T) swabs are invasive and require trained medical staff for reliable sampling. In addition, it has been shown that PCR is more sensitive as compared to antigen-based tests. Here we describe the analytical and clinical evaluation of our in-house RNA extraction-free saliva-based molecular assay for the detection of SARS-CoV-2. Analytical sensitivity of the test was equal to the sensitivity obtained in other Dutch diagnostic laboratories that process NP/T swabs. In this study, 955 individuals participated and provided NP/T swabs for routine molecular analysis (with RNA extraction) and saliva for comparison. Our RT-qPCR resulted in a sensitivity of 82,86% and a specificity of 98,94% compared to the gold standard. A false-negative ratio of 1,9% was found. The SARS-CoV-2 detection workflow described here enables easy, economical, and reliable saliva processing, useful for repeated testing of individuals.

Deficiency of either P-glycoprotein or breast cancer resistance protein protect against acute kidney injury.

The kidney has a high capacity to regenerate after ischemic injury via several mechanisms, one of which involves bone marrow-derived (stem) cells. The ATP binding cassette transporters, P-glycoprotein and breast cancer resistance protein, are determinants for the enriched stem and progenitor cell fraction in bone marrow. Because they are upregulated after acute kidney injury, we hypothesized that both efflux pumps may play a role in protecting against renal injury. Surprisingly, transporter-deficient mice were protected against ischemia-induced renal injury. To further study this, bone marrow from irradiated wild-type mice was reconstituted by bone marrow from wild-type, P-glycoprotein- or breast cancer resistance protein-deficient mice. Four weeks later, kidney injury was induced and its function evaluated. Significantly more bone marrow-derived cells were detected in kidneys grafted with transporter-deficient bone marrow. A gender mismatch study suggested that cell fusion of resident tubular cells with bone marrow cells was unlikely. Renal function analyses indicated an absence of renal damage following ischemia-reperfusion in animals transplanted with transporter-deficient bone marrow. When wild-type bone marrow was transplanted in breast cancer resistance protein-deficient mice this protection is lost. Furthermore, we demonstrate that transporter-deficient bone marrow contained significantly more monocytes, granulocytes, and early outgrowth endothelial progenitor cells.

Testosterone increases urinary calcium excretion and inhibits expression of renal calcium transport proteins.

Although gender differences in the renal handling of calcium have been reported, the overall contribution of androgens to these differences remains uncertain. We determined here whether testosterone affects active renal calcium reabsorption by regulating calcium transport proteins. Male mice had higher urinary calcium excretion than female mice and their renal calcium transporters were expressed at a lower level. We also found that orchidectomized mice excreted less calcium in their urine than sham-operated control mice and that the hypocalciuria was normalized after testosterone replacement. Androgen deficiency increased the abundance of the renal mRNA and protein of both the luminal transient receptor potential vanilloid-subtype 5 (TRPV5) and intracellular calbindin-D(28K) transporters, which in turn were suppressed by testosterone treatment. There were no significant differences in serum estrogen, parathyroid hormone, or 1,25-dihydroxyvitamin D3 levels between control and orchidectomized mice with or without testosterone. Moreover, incubation of primary rabbit connecting tubule and cortical collecting duct cells with a nonaromatizable androgen, dihydrotestosterone, reduced transcellular calcium transport. Thus, our study shows that gender differences in renal calcium handling are, in part, mediated by the inhibitory actions of androgens on TRPV5-mediated active renal calcium transport.

Activation of the Ca2+-sensing receptor stimulates the activity of the epithelial Ca2+ channel TRPV5.

The extracellular Ca(2+)-sensing receptor (CaR) is a key-player in plasma Ca(2+) homeostasis. It is essentially expressed in the parathyroid glands and along the kidney nephron. The distal convoluted tubules (DCT) and connecting tubules (CNT) in the kidney are involved in active Ca(2+) reabsorption, but the function of the CaR has remained unclear in these segments. Here, the Ca(2+)-selective Transient Receptor Potential Vanilloid-subtype 5 channel (TRPV5) determines active Ca(2+) reabsorption by forming the apical entry gate. In this study we show that the CaR and TRPV5 co-localize at the luminal membrane of DCT/CNT. Furthermore, by patch-clamp and Fura-2-ratiometric measurements we demonstrate that activation of the CaR leads to elevated TRPV5-mediated currents and increases intracellular Ca(2+) concentrations in cells co-expressing TRPV5 and CaR. Activation of CaR initiated a signaling cascade that activated phorbol-12-myristate-13-acetate (PMA)-insensitive protein kinase C (PKC) isoforms. Importantly, mutation of two putative PKC phosphorylation sites, S299 and S654, in TRPV5 prevented the stimulatory effect of CaR activation on channel activity, as did a dominant negative CaR construct, CaR(R185Q). Interestingly, the activity of TRPV6, TRPV5' closest homologue, was not affected by the activated CaR. We conclude that activation of the CaR stimulates TRPV5-mediated Ca(2+) influx via a PMA-insensitive PKC isoform pathway.

Conditional fast expression and function of multimeric TRPV5 channels using Shield-1.

A recently described novel controllable method to regulate protein expression is based on a mutated FK506-binding protein-12 (mtFKBP) that is unstable and rapidly degraded in mammalian cells. This instability can be conferred to other proteins directly fused to mtFKBP. Binding of a synthetic cell-permeant ligand (Shield-1) to mtFKBP reverses the instability, allowing conditional expression of mtFKBP-fused proteins. We adapted this strategy to study multimeric plasma membrane proteins using the ion channel TRPV5 as model protein. mtFKBP-TRPV5 forms functional ion channels and its expression can be controlled in a time- and dose-dependent fashion using Shield-1. Moreover, in the presence of Shield-1, mtFKBP-TRPV5 formed heteromultimeric channels with untagged TRPV5, which were codegraded upon washout of Shield-1, providing a strategy to study multimeric plasma membrane protein complexes without the need to destabilize all individual subunits.

Intracellular signal transduction by the extracellular calcium-sensing receptor of Xenopus melanotrope cells.

The extracellular calcium-sensing receptor (CaR) is expressed in various types of endocrine pituitary cell, but the intracellular mechanism this G protein-coupled receptor uses in these cells is not known. In the present study we investigated possible intracellular signal transduction pathway(s) utilized by the CaR of the endocrine melanotrope cells in the intermediate pituitary lobe of the South African-clawed toad Xenopus laevis. For this purpose, the effects of various pharmacological agents on CaR-evoked secretion of radiolabeled secretory peptides from cultured melanotrope cells were assessed. CaR-evoked secretion, induced by the potent CaR agonist L-phenylalanine (L-Phe), could not be inhibited by cholera toxin, nor by NPC-15437 and PMA, indicating that neither G(s)/PKA nor G(q)/PKC pathways are involved. However, pertussis toxin (G(i/o) protein inhibitor), genistein (inhibitor of PTKs), wortmannin/LY-294002 (PI3-K inhibitor) and U-0126 (inhibitor of extracellular signal-regulated kinase, ERK) all substantially inhibited CaR-evoked secretion, indicating that the Xenopus melanotrope cell possesses a PI3-K/MAPK system that plays some role in CaR-signaling. Since no direct effect of L-Phe on ERK phosphorylation could be shown it is concluded that CaR must act primarily through another, still unknown, signaling pathway in Xenopus melanotropes. Our results indicate that the PI3-K/MAPK system has a facilitating effect on CaR-induced secretion, possibly by sensitizing the CaR.

Identification of Nipsnap1 as a novel auxiliary protein inhibiting TRPV6 activity.

The transient receptor potential vanilloid channels 5 and 6 (TRPV5/6) are the most Ca(2+)-selective channels within the TRP superfamily of ion channels. These epithelial Ca(2+) channels are regulated at different intra- and extracellular sites by the feedback response of Ca(2+) itself, calciotropic hormones, and by TRPV5/6-associated proteins. In the present study, bioinformatics was used to search for novel TRPV5/6-associated genes. By including pull-down assays and functional analysis, Nipsnap1-a hitherto functionally uncharacterized globular protein-was identified as a novel factor involved in the regulation of TRPV6. Electrophysiological recordings revealed that Nipsnap1 abolishes TRPV6 currents. Subsequent biotinylation assays showed that TRPV6 plasma membrane expression did not change in the presence of Nipsnap1, suggesting that TRPV6 inhibition by Nipsnap1 is independently regulated from reduced cell surface channel expression. In addition, semi-quantitative reverse transcriptase PCR and immunohistochemical labeling of Nipsnap1 indicated that Nipsnap1 is expressed in mouse intestinal tissues-where TRPV6 is predominantly expressed-but that it does not co-localize with TRPV5 in the kidney. In conclusion, this study presents the first physiological function of Nipsnap1 as an associated protein inhibiting TRPV6 activity that possibly exerts its effect directly at the plasma membrane.