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Species of the genus Phaeohelotium (Leotiomycetes: Helotiaceae) are cup fungi that grow on decaying wood, leaves, litter, and directly on soil. Northern Hemisphere species are primarily found on litter and wood, whereas in the Southern Hemisphere the genus includes a mix of saprotrophs as well as taxa that grow on soil in association with ectomycorrhizal trees. The diversity of this genus has not been fully explored in southern South America. Here we describe two species from Chile, Phaeohelotium maiusaurantium sp. nov. and Ph. pallidum sp. nov., found on soil in Patagonian Nothofagaceae-dominated forests. We present macro- and micromorphological descriptions, illustrations, and molecular phylogenetic analyses. The two new species are placed in Phaeohelotium with high support in our 15-locus phylogeny as well as phylogenetic reconstructions based on the internal transcribed spacer (ITS) region of the nuclear ribosomal RNA gene. Our ITS phylogeny places both Ph. maiusaurantium and Ph. pallidum in a well-supported subclade that includes ectomycorrhizal root tip samples from Australasia. Similar species can be separated from these new taxa based on morphological characteristics, biogeography, substrate, and sequence data. In addition, two unnamed species from Chilean Nothofagaceae forests (Phaeohelotium sp. 1 and Phaeohelotium sp. 2) are documented from scant collections and sequence data and await description until more material becomes available.

International audience; The reactive oxygen species (ROS)-producing enzyme NADPH oxidase (NOX) was first identified in the membrane of phagocytic cells. For many years, its only known role was in immune defense, where its ROS production leads to the destruction of pathogens by the immune cells. NOX from phagocytes catalyzes, via one-electron trans-membrane transfer to molecular oxygen, the production of the superoxide anion. Over the years, six human homologs of the catalytic subunit of the phagocyte NADPH oxidase were found: NOX1, NOX3, NOX4, NOX5, DUOX1, and DUOX2. Together with the NOX2/gp91$^{phox}$ component present in the phagocyte NADPH oxidase assembly itself, the homologs are now referred to as the NOX family of NADPH oxidases. NOX are complex multidomain proteins with varying requirements for assembly with combinations of other proteins for activity. The recent structural insights acquired on both prokaryotic and eukaryotic NOX open new perspectives for the understanding of the molecular mechanisms inherent to NOX regulation and ROS production (superoxide or hydrogen peroxide). This new structural information will certainly inform new investigations of human disease. As specialized ROS producers, NOX enzymes participate in numerous crucial physiological processes, including host defense, the post-translational processing of proteins, cellular signaling, regulation of gene expression, and cell differentiation. These diversities of physiological context will be discussed in this review. We also discuss NOX misregulation, which can contribute to a wide range of severe pathologies, such as atherosclerosis, hypertension, diabetic nephropathy, lung fibrosis, cancer, or neurodegenerative diseases, giving this family of membrane proteins a strong therapeutic interest.

Voltage-gated proton channels open appropriately in myriad physiological situations because their gating is powerfully modulated by both pHo and pHi. Cherny et al. serendipitously identify a histidine at the inner end of the S3 helix that is required for the response to pHi., We recently identified a voltage-gated proton channel gene in the snail Helisoma trivolvis, HtHV1, and determined its electrophysiological properties. Consistent with early studies of proton currents in snail neurons, HtHV1 opens rapidly, but it unexpectedly exhibits uniquely defective sensitivity to intracellular pH (pHi). The H+ conductance (gH)-V relationship in the voltage-gated proton channel (HV1) from other species shifts 40 mV when either pHi or pHo (extracellular pH) is changed by 1 unit. This property, called ΔpH-dependent gating, is crucial to the functions of HV1 in many species and in numerous human tissues. The HtHV1 channel exhibits normal pHo dependence but anomalously weak pHi dependence. In this study, we show that a single point mutation in human hHV1—changing His168 to Gln168, the corresponding residue in HtHV1—compromises the pHi dependence of gating in the human channel so that it recapitulates the HtHV1 response. This location was previously identified as a contributor to the rapid gating kinetics of HV1 in Strongylocentrotus purpuratus. His168 mutation in human HV1 accelerates activation but accounts for only a fraction of the species difference. H168Q, H168S, or H168T mutants exhibit normal pHo dependence, but changing pHi shifts the gH-V relationship on average by

Voltage-gated proton currents were first discovered in snail neurons. Thomas et al. identify a snail proton channel gene that codes for a rapidly activating proton channel that differs from other proton channels, in particular by its low sensitivity to intracellular pH., Voltage-gated proton channels, HV1, were first reported in Helix aspersa snail neurons. These H+ channels open very rapidly, two to three orders of magnitude faster than mammalian HV1. Here we identify an HV1 gene in the snail Helisoma trivolvis and verify protein level expression by Western blotting of H. trivolvis brain lysate. Expressed in mammalian cells, HtHV1 currents in most respects resemble those described in other snails, including rapid activation, 476 times faster than hHV1 (human) at pHo 7, between 50 and 90 mV. In contrast to most HV1, activation of HtHV1 is exponential, suggesting first-order kinetics. However, the large gating charge of ∼5.5 e0 suggests that HtHV1 functions as a dimer, evidently with highly cooperative gating. HtHV1 opening is exquisitely sensitive to pHo, whereas closing is nearly independent of pHo. Zn2+ and Cd2+ inhibit HtHV1 currents in the micromolar range, slowing activation, shifting the proton conductance–voltage (gH-V) relationship to more positive potentials, and lowering the maximum conductance. This is consistent with HtHV1 possessing three of the four amino acids that coordinate Zn2+ in mammalian HV1. All known HV1 exhibit ΔpH-dependent gating that results in a 40-mV shift of the gH-V relationship for a unit change in either pHo or pHi. This property is crucial for all the functions of HV1 in many species and numerous human cells. The HtHV1 channel exhibits normal or supernormal pHo dependence, but weak pHi dependence. Under favorable conditions, this might result in the HtHV1 channel conducting inward currents and perhaps mediating a proton action potential. The anomalous ΔpH-dependent gating of HtHV1 channels suggests a structural basis for this important property, which is further explored in this issue (Cherny et al. 2018. J. Gen. Physiol. https://doi.org/10.1085/jgp.201711968).

Background: Trimodality bladder preservation therapy (BPT) in muscle invasive bladder cancer (MIBC) includes a maximal transurethral resection followed by concurrent chemoradiotherapy as an alternative to radical cystectomy (RC) in appropriately selected patients, or as a treatment option in non-cystectomy candidates. Several chemotherapy regimens can be used in BPT, but little is known about current practice patterns. Objective: To describe utilization patterns of BPT and associated survival outcomes in MIBC. Methods: Data were collected from the Retrospective International Study of Cancers of the UrothelialTract (RISC), a database of 3,024 consecutive patients from 29 international academic centers from 2005 to 2013. Patients with clinical T2-T4aN0M0 urothelial cancer of the bladder were included. Results: 265 patients received BPT. Compared with the 1,447 patients who received RC, BPT patients were older, had poorer performance status, and had more comorbidities (p < 0.01 for all). Median overall survival (OS) was similar for patients treated with curative radiation doses in BPT and patients treated with RC (41 vs 46 months, p = 0.33, respectively). 45% of BPT patients received concurrent chemotherapy with radiation. The most common regimens included cisplatin alone (23%), carboplatin alone (22%), gemcitabine alone (10%), paclitaxel alone (9%), and 5-FU+mitomycin (5%). There were no significant differences in survival among chemotherapy regimens. Only 10 patients (4% of BPT patients) underwent salvage cystectomy. Conclusions: In clinical practice, BPT patients have similar survival to RC patients when treated with curative radiotherapy doses. Choice of concurrent chemotherapy regimen varied widely with no clear standard. Salvage cystectomy is rarely performed. Continued research is needed on the comparative effectiveness among BPT and RC, and among chemotherapy regimens in BPT. © 2016 - IOS Press and the authors. All rights reserved.

Voltage-gated proton channels are considered completely selective for protons — no evidence exists for the permeation of ions other than H+. A study of the underlying mechanism of the high selectivity of the human voltage-gated proton channel reveals an Asp112 residue as the selectivity filter. When this residue was changed to a neutral amino acid, the mutant channel lost proton selectivity and either became anion-selective or did not conduct any ions. The ion selectivity of pumps and channels is central to their ability to perform a multitude of functions. Here we investigate the mechanism of the extraordinary selectivity of the human voltage-gated proton channel1, HV1 (also known as HVCN1). This selectivity is essential to its ability to regulate reactive oxygen species production by leukocytes2,3,4, histamine secretion by basophils5, sperm capacitation6, and airway pH7. The most selective ion channel known, HV1 shows no detectable permeability to other ions1. Opposing classes of selectivity mechanisms postulate that (1) a titratable amino acid residue in the permeation pathway imparts proton selectivity1,8,9,10,11, or (2) water molecules ‘frozen’ in a narrow pore conduct protons while excluding other ions12. Here we identify aspartate 112 as a crucial component of the selectivity filter of HV1. When a neutral amino acid replaced Asp 112, the mutant channel lost proton specificity and became anion-selective or did not conduct. Only the glutamate mutant remained proton-specific. Mutation of the nearby Asp 185 did not impair proton selectivity, indicating that Asp 112 has a unique role. Although histidine shuttles protons in other proteins, when histidine or lysine replaced Asp 112, the mutant channel was still anion-permeable. Evidently, the proton specificity of HV1 requires an acidic group at the selectivity filter.

We previously characterized a H + transport pathway in medullary thick ascending limb nephron segments that when activated stimulated the production of superoxide by nicotinamide adenine dinucleotide phosphate oxidase. Importantly, the activity of this pathway was greater in Dahl salt-sensitive rats than salt-resistant (SS.13 BN ) rats, and superoxide production was enhanced in low Na + media. The goal of this study was to determine the molecular identity of this pathway and its relationship to Na + . We hypothesized that the voltage-gated proton channel, HV1, was the source of superoxide-stimulating H + currents. To test this hypothesis, we developed HV1 −/− null mutant rats on the Dahl salt-sensitive rat genetic background using zinc-finger nuclease gene targeting. HV1 could be detected in medullary thick limb from wild-type rats. Intracellular acidification using an NH 4 Cl prepulse in 0 sodium/BaCl 2 containing media resulted in superoxide production in thick limb from wild-type but not HV1 −/− rats ( P I 0.005 versus 0.002 U/s, P =0.046, respectively). Superoxide production was enhanced by low intracellular sodium (−/− rats compared with wild-type Dahl salt-sensitive rats. We conclude that HV1 is expressed in medullary thick ascending limb and promotes superoxide production in this segment when intracellular Na + is low. HV1 contributes to the development of hypertension and renal disease in Dahl salt-sensitive rats.

SummaryNADPH oxidases (Nox) are a primary source of reactive oxygen species (ROS), which function in normal physiology and, when overproduced, in pathophysiology. Recent studies using mice deficient in Nox2 identify this isoform as a novel target against Nox2-implicated inflammatory diseases. Nox2 activation depends on the binding of the proline-rich domain of its heterodimeric partner p22phox to p47phox. A high-throughput screen that monitored this interaction via fluorescence polarization identified ebselen and several of its analogs as inhibitors. Medicinal chemistry was performed to explore structure-activity relationships and to optimize potency. Ebselen and analogs potently inhibited Nox1 and Nox2 activity but were less effective against other isoforms. Ebselen also blocked translocation of p47phox to neutrophil membranes. Thus, ebselen and its analogs represent a class of compounds that inhibit ROS generation by interrupting the assembly of Nox2-activating regulatory subunits.

Mice fed a semipurified, vitamin A-deficient diet (A- mice) and control animals fed the same diet with added retinyl acetate (A+ mice) were used to investigate the effect of vitamin A deficiency on primary immunoglobulin responses to protein antigens. At age 6 weeks, A- mice had serum retinol concentrations that were 46% of A+ controls. When immunized with a single antigen dose, these mice produced an antigen-specific IgM response equivalent to controls, but their IgG1 and IgG3 responses were sharply diminished (less than 30% of A+ controls). At age 8 weeks, A- mice had 20% of A+ serum retinol concentrations and less than 17% of A+ liver retinyl palmitate levels. Responding to a single antigen dose, A- mice produced approximately equal to 70% as much IgM as A+ controls. Their IgG1 response was less than 30% and their IgG3 response less than 3% of A+ controls. The IgG1 response kinetics were identical in A- and A+ mice. Diminished serum antibody responses in A- mice were attributable to fewer immunoglobulin-secreting plasma cells rather than to a decline in IgM or IgG secretion rate per cell. Total serum IgG3 levels, irrespective of antigen specificity, were slightly elevated in A- mice compared to A+ controls. The inefficient clonal expansion of responding B lymphocytes and contrasting impairment of IgM and IgG responses observed in vitamin A-deficient mice are discussed with respect to a possible helper/inducer-T-lymphocyte defect.

The voltage-gated proton channel (Hv1) of leukocytes, basophils, airway epithelium, and spermatozoa is extremely selective. Hv1 presents structural and functional similarities to the voltage-sensor domain (VSD) of voltage-gated potassium (Kv) and sodium (Nav) channels. Hv1 and the VSDs of Kv and Nav sense changes in membrane potentials and contain four α-helical segments as well as conserved arginine residues on the fourth helix. Although the electrophysiological features of Hv1 are well characterized, the molecular mechanism of proton conduction is unknown. This is largely because an experimentally-determined structure of Hv1 is not available. An alternative source of structural information is homology modeling, whereby a model of Hv1 can be constructed using the atomic structures of the VSDs of Kv and Nav as templates. However, since the sequence identity between Hv1 and templates is below 20%, a critical assessment of generated models is essential. Here we present a homology modeling-simulation strategy using alternative sequence alignments to construct, assess, and validate alternative homology models. Comparison of a range of structural properties between the models and templates is used to guide the selection of an optimal model for Hv1. This method represents a generalized strategy that can be applied to other membrane proteins that lack high sequence identity to their templates.

Extraordinary selectivity is crucial to all proton conducting molecules, including the human voltage gated proton channel, hHV1, because [H+] is minuscule. Here we use "selectivity filter scanning" to elucidate the molecular requirements for proton specific conduction in hHV1. Asp112, in the middle of the S1 transmembrane helix, is essential to the WT channel selectivity [Musset et al., 2011. Nature 480:273-277]. We neutralized Asp112 by mutating it to Ala (D112A), then introduced Asp at each position along S1 from 108 to 118, searching for "second site suppressor" activity. All mutants except for D112A/V109D lacked even the anion conduction exhibited by D112A. Proton specific conduction was restored with Asp or Glu at position 116. The D112V/V116D channel resembled WT in selectivity, kinetics, and ΔpH dependent gating. Both R211H and R211H/D112V/V116D were inhibited by internally applied Zn2+ when the channel was open, indicating similar S4 accessibility. At position 109 Asp allowed anion permeation in combination with D112A, but did not rescue function in the nonconducting D112V mutant, indicating that selectivity is established external to the constriction at Phe150. The three positions (109, 112, 116) that permit conduction all line the pore in our homology model, delineating the conduction pathway. Evidently, proton selective conduction requires a carboxyl group to face the pore at a constriction in the external vestibule. Molecular dynamics studies indicate reorganization of ionic networks in response to mutations and suggest that the distribution of charged groups in the external vestibule modulates charge selectivity. That the selectivity filter functions in a new location helps define local environmental features that produce proton selective conduction.

Sequence and phylogenetic analysis of voltage-gated ion channels has famously resulted in the discovery of novel ion channels in previously unexplored phyla, and has provided insight into the molecular underpinnings of ion channel function. Because of their crucial role in human health, most current evolutionary analysis has quite reasonably centered on ‘traditional’ pore-forming channels (e.g., Yu, et al, 2005, Pharmacol. Rev., 57:387-395, among many others). Earlier phylogenetic analyses of voltage sensor domain (VSD) modules (e.g., Komanovics et al, 2002, FASEB J., 16:1623-1629, among others) were undertaken before the discovery and characterization of the genes for voltage sensitive phosphatases (VSP) and voltage gated proton channels (HV1), which contain VSDs but do not contain traditional ion pores. We recently reported two new discoveries: in one line of inquiry we found and characterized a dinoflagellate HV1, supporting the prediction of its existence in bioluminescent dinoflagellates by Fogel and Hastings in 1972 (PNAS, 3:690-693); and in the other we uncovered the universal selectivity filter of HV1's. Both of these studies were informed by large-scale sequence and phylogenetic analyses that focused on homologs of VSDs themselves, separate from their N- or C-terminal appendages. Here we present a full sequence and phylogenetic analysis that updates earlier work and reveals that VSDs may have taken a different evolutionary path from associated ion channel pore domains, which has mechanistic and physiological implications.