Dysregulation of the WNK4-SPAK/OSR1 pathway has a minor effect on baseline NKCC2 phosphorylation.

The with-no-lysine kinase 4 (WNK4)-sterile 20/SPS-1-related proline/alanine-rich kinase (SPAK)/oxidative stress-responsive kinase 1 (OSR1) pathway mediates activating phosphorylation of the furosemide-sensitive Na+-K+-2Cl- cotransporter (NKCC2) and the thiazide-sensitive NaCl cotransporter (NCC). The commonly used pT96/pT101-pNKCC2 antibody cross-reacts with pT53-NCC in mice on the C57BL/6 background due to a five amino acid deletion. We generated a new C57BL/6-specific pNKCC2 antibody (anti-pT96-NKCC2) and tested the hypothesis that the WNK4-SPAK/OSR1 pathway strongly regulates the phosphorylation of NCC but not NKCC2. In C57BL/6 mice, anti-pT96-NKCC2 detected pNKCC2 and did not cross-react with NCC. Abundances of pT96-NKCC2 and pT53-NCC were evaluated in Wnk4-/-, Osr1-/-, Spak-/-, and Osr1-/-/Spak-/- mice and in several models of the disease familial hyperkalemic hypertension (FHHt) in which the CUL3-KLHL3 ubiquitin ligase complex that promotes WNK4 degradation is dysregulated (Cul3+/-/Δ9, Klhl3-/-, and Klhl3R528H/R528H). All mice were on the C57BL/6 background. In Wnk4-/- mice, pT53-NCC was almost absent but pT96-NKCC2 was only slightly lower. pT53-NCC was almost absent in Spak-/- and Osr1-/-/Spak-/- mice, but pT96-NKCC2 abundance did not differ from controls. pT96-NKCC2/total NKCC2 was slightly lower in Osr1-/- and Osr1-/-/Spak-/- mice. WNK4 expression colocalized not only with NCC but also with NKCC2 in Klhl3-/- mice, but pT96-NKCC2 abundance was unchanged. Consistent with this, furosemide-induced urinary Na+ excretion following thiazide treatment was similar between Klhl3-/- and controls. pT96-NKCC2 abundance was also unchanged in the other FHHt mouse models. Our data show that disruption of the WNK4-SPAK/OSR1 pathway only mildly affects NKCC2 phosphorylation, suggesting a role for other kinases in NKCC2 activation. In FHHt models NKCC2 phosphorylation is unchanged despite higher WNK4 abundance, explaining the thiazide sensitivity of FHHt.NEW & NOTEWORTHY The renal cation cotransporters NCC and NKCC2 are activated following phosphorylation mediated by the WNK4-SPAK/OSR1 pathway. While disruption of this pathway strongly affects NCC activity, effects on NKCC2 activity are unclear since the commonly used phospho-NKCC2 antibody was recently reported to cross-react with phospho-NCC in mice on the C57BL/6 background. Using a new phospho-NKCC2 antibody specific for C57BL/6, we show that inhibition or activation of the WNK4-SPAK/OSR1 pathway in mice only mildly affects NKCC2 phosphorylation.

Cysteine-rich peptides: From bioactivity to bioinsecticide applications.

Greater levels of insect resistance and constraints on the use of current pesticides have recently led to increased crop losses in agricultural production. Further, the health and environmental impacts of pesticides now restrict their application. Biologics based on peptides are gaining popularity as efficient crop protection agents with low environmental toxicity. Cysteine-rich peptides (whether originated from venoms or plant defense substances) are chemically stable and effective as insecticides in agricultural applications. Cysteine-rich peptides fulfill the stability and efficacy requirements for commercial uses and provide an environmentally benign alternative to small-molecule insecticides. In this article, cysteine-rich insecticidal peptide classes identified from plants and venoms will be highlighted, focusing on their structural stability, bioactivity and production.

Insights into the synthesis strategies of plant-derived cyclotides.

Cyclotides are plant peptides characterized with a head-to-tail cyclized backbone and three interlocking disulfide bonds, known as a cyclic cysteine knot. Despite the variations in cyclotides peptide sequences, this core structure is conserved, underlying their most useful feature: stability against thermal and chemical breakdown. Cyclotides are the only natural peptides known to date that are orally bioavailable and able to cross cell membranes. Cyclotides also display bioactivities that have been exploited and expanded to develop as potential therapeutic reagents for a wide range of conditions (e.g., HIV, inflammatory conditions, multiple sclerosis, etc.). As such, in vitro production of cyclotides is of the utmost importance since it could assist further research on this peptide class, specifically the structure-activity relationship and its mechanism of action. The information obtained could be utilized to assist drug development and optimization. Here, we discuss several strategies for the synthesis of cyclotides using both chemical and biological routes.

Cullin 3 mutant causing familial hyperkalemic hypertension lacks normal activity in the kidney.

Mutations in the ubiquitin ligase scaffold protein cullin 3 (CUL3) cause the disease familial hyperkalemic hypertension (FHHt). We recently reported that in the kidney, aberrant mutant CUL3 (CUL3-Δ9) activity lowers the abundance of CUL3-Δ9 and Kelch-like 3, the CUL3 substrate adaptor for with-no-lysine kinase 4 (WNK4) and that this is mechanistically important. However, whether CUL3-Δ9 exerts additional effects on other targets that may alter renal function is unclear. Here, we sought to determine 1) whether CUL3-Δ9 expression can rescue the phenotype of renal tubule-specific Cul3 knockout mice, and 2) whether CUL3-Δ9 expression affects other CUL3 substrates. Using an inducible renal tubule-specific system, we studied two CUL3-Δ9-expressing mouse models: Cul3 knockout (Cul3-/-/Δ9) and Cul3 heterozygous background (Cul3+/-/Δ9, FHHt model). The effects of CUL3-Δ9 in these mice were compared with Cul3-/- and Cul3+/- mice. Similar to Cul3-/- mice, Cul3-/-/Δ9 mice displayed polyuria with loss of aquaporin 2 and collecting duct injury; proximal tubule injury also occurred. CUL3-Δ9 did not promote degradation of two CUL3 targets that accumulate in the Cul3-/- kidney: high-molecular-weight (HMW) cyclin E and NAD(P)H:quinone oxidoreductase 1 (NQO1) [a surrogate for the CUL3-Kelch-like ECH-associated protein 1 (KEAP1) substrate nuclear factor erythroid-2-related factor 2]. Since CUL3-Δ9 expression cannot rescue the Cul3-/- phenotype, our data suggest that CUL3-Δ9 cannot normally function in ubiquitin ligase complexes. In Cul3+/-/Δ9 mice, KEAP1 abundance did not differ but NQO1 abundance was higher, suggesting adaptor sequestration by CUL3-Δ9 in vivo. Together, our results provide evidence that in the kidney, CUL3-Δ9 completely lacks normal activity and can trap CUL3 substrate adaptors in inactive complexes.NEW & NOTEWORTHY CUL3 mutation (CUL3-Δ9) causes familial hyperkalemic hypertension (FHHt) by reducing adaptor KLHL3, impairing substrate WNK4 degradation. Whether CUL3-Δ9 affects other targets in kidneys remains unclear. We found that CUL3-Δ9 cannot degrade two CUL3 targets, cyclin E and nuclear factor erythroid-2-related factor 2 (NRF2; using a surrogate marker NQO1), or rescue injury or polyuria caused by Cul3 disruption. In an FHHt model, CUL3-Δ9 impaired NRF2 degradation without reduction of its adaptor KEAP1. Our data provide additional insights into CUL3-Δ9 function in the kidney.

Validation of Home Visual Acuity Tests for Telehealth in the COVID-19 Era.

Importance: Visual acuity (VA) is one of the most important clinical data points in ophthalmology. However, few options for validated at-home VA assessments are currently available. Objective: To validate 3 at-home visual acuity tests in comparison with in-office visual acuity. Design, Setting, and Participants: Between July 2020 and April 2021, eligible participants with VA of 20/200 or better were recruited from 4 university-based ophthalmology clinics (comprehensive, cornea, glaucoma, and retina clinics). Participants were prospectively randomized to self-administer 2 of 3 at-home VA tests (printed chart, mobile phone app, and website) within 3 days before their standard-of-care clinic visit. Participants completed a survey assessing usability of the at-home tests. At the clinic visit, best-corrected Snellen distance acuity was measured as the reference standard. Main Outcomes and Measures: The at-home VA test results were compared with the in-office VA test results using paired and unpaired t tests, Pearson correlation coefficients, analysis of variance, χ2 tests, and Cohen κ agreement. The sensitivity, specificity, positive predictive value, and negative predictive value of each at-home test were calculated to detect significant VA changes (≥0.2 logMAR) from the in-office baseline. Results: A total of 121 participants with a mean (SD) age of 63.8 (13.0) years completed the study. The mean in-office VA was 0.11 logMAR (Snellen equivalent 20/25) with similar numbers of participants from the 4 clinics. Mean difference (logMAR) between the at-home test and in-office acuity was -0.07 (95% CI, -0.10 to -0.04) for the printed chart, -0.12 (95% CI, -0.15 to -0.09) for the mobile phone app, and -0.13 (95% CI, -0.16 to -0.10) for the website test. The Pearson correlation coefficient for the printed chart was 0.72 (95% CI, 0.62-0.79), mobile phone app was 0.58 (95% CI, 0.46-0.69), and website test was 0.64 (95% CI, 0.53-0.73). Conclusions and Relevance: The 3 at-home VA test results (printed chart, mobile phone app, and website) appeared comparable within 1 line to in-office VA measurements. Older participants were more likely to have limited access to digital tools. Further development and validation of at-home VA testing modalities is needed with the expansion of teleophthalmology care.

Asymmetric dimerization of adenosine deaminase acting on RNA facilitates substrate recognition.

Adenosine deaminases acting on RNA (ADARs) are enzymes that convert adenosine to inosine in duplex RNA, a modification that exhibits a multitude of effects on RNA structure and function. Recent studies have identified ADAR1 as a potential cancer therapeutic target. ADARs are also important in the development of directed RNA editing therapeutics. A comprehensive understanding of the molecular mechanism of the ADAR reaction will advance efforts to develop ADAR inhibitors and new tools for directed RNA editing. Here we report the X-ray crystal structure of a fragment of human ADAR2 comprising its deaminase domain and double stranded RNA binding domain 2 (dsRBD2) bound to an RNA duplex as an asymmetric homodimer. We identified a highly conserved ADAR dimerization interface and validated the importance of these sequence elements on dimer formation via gel mobility shift assays and size exclusion chromatography. We also show that mutation in the dimerization interface inhibits editing in an RNA substrate-dependent manner for both ADAR1 and ADAR2.

Defect mapping in pipes by ultrasonic wavefield cross-correlation: A synthetic verification.

This work presents a reverse-time imaging technique by cross-correlating the forward wavefield with the reverse wavefield for the detection, localization, and sizing of defects in pipelines. The presented technique allows to capture the wavefield reflectivity at the places of ultrasonic wave scattering and reflections. Thus, the method is suitable for detecting pipe defects of either point-like or finite-size types using data from a pulse-echo setup. By using synthetic data generated by 3D spectral element pipe models, we show that the 3D wavefield cross-correlation imaging is capable in the case of cylindrical guided ultrasonic waves. With a ring setup of transducers, we analyze the imaging results obtained from the synthetic single-transducer and all-transducer firings. The presented pipe flaw imaging method is straightforward to carry out using a suitable wave equation solver. Also, the method does not suffer from long iterative runs and numerical convergence issues commonly connected with imaging methods based on either deterministic optimization or statistical inference. The imaging procedure can be fully baseline-free by performing data processing to remove direct arrivals from the ultrasound data.

Ultrasonic wavefield inversion and migration in complex heterogeneous structures: 2D numerical imaging and nondestructive testing experiments.

Delaminations, cracks and other defects in engineered structures often lie close to the theoretical resolution limit for ultrasonic waves. While ultrasonic waveform tomography has succeeded in detecting such features, recovery is difficult because it requires computationally expensive high-frequency numerical wave simulations and an accurate understanding of large-scale background variations of the engineered structure. Without such knowledge, small defects may be incorrectly imaged or go undetected altogether. To reduce computational cost and improve detection of small defects, a useful approach is to divide the waveform tomography procedure into two steps: first, a low-frequency model-building step aimed at recovering background structure, and second, a high-frequency imaging step targeting defects. The first is naturally formulated as waveform inversion for wavespeed parameters and the second as time reversal migration for reflectivity. Through synthetic test cases, we show that the two-step workflow appears more promising in most cases than a single-step inversion. In particular, we find that new workflow succeeds in the challenging scenario where the defect lies along preexisting layer interface in a composite bridge deck and in related experiments involving noisy data or inaccurate source parameters.

Photodissociation of ozone in the Hartley band: Product state and angular distributions.

Product state properties from the photodissociation of ozone in the ultraviolet Hartley band are investigated by trajectory surface-hopping calculations. The diabatic B and R state potential energy and coupling surfaces of Schinke and McBane [J. Chem. Phys. 132, 044305 (2010)] are employed. The properties computed include rotational and vibrational distributions in both the singlet and triplet channels, the total internal energy distribution in the triplet channel, and the photodissociation anisotropy parameter ß in the singlet channel. A method for computing ß from trajectories computed in internal Jacobi coordinates is described. In the singlet channel, the vibrational distribution is in good agreement with the experimental results. The observed increase in ß with increasing photolysis wavelength is reproduced by the calculations and is attributed to the effects of the bending potential on the B state late in the fragmentation. The computed ß values are too high with respect to experiment, and the peaks j(max) of the singlet-channel rotational distributions are too low; these discrepancies are attributed to a too steep bending potential at long O-O distances. In the triplet channel, the main part of the internal energy distribution is described well by the calculations, although the detailed structures observed in the experiment are not reproduced. The experimental rotational distributions are well reproduced, although the maxima appear at slightly too high j. The triplet state product energy distributions are shown to depend largely on the distribution of hopping points onto the R state surface. A Landau-Zener model constructed as a function of the O(2) bond distance provides a good physical description of the two-state dynamics. The high internal energy O(2) products that cannot be attributed to the excitation of the Herzberg states remain unexplained.