Identification of a drug binding pocket in TMEM16F calcium-activated ion channel and lipid scramblase.

The dual functions of TMEM16F as Ca2+-activated ion channel and lipid scramblase raise intriguing questions regarding their molecular basis. Intrigued by the ability of the FDA-approved drug niclosamide to inhibit TMEM16F-dependent syncytia formation induced by SARS-CoV-2, we examined cryo-EM structures of TMEM16F with or without bound niclosamide or 1PBC, a known blocker of TMEM16A Ca2+-activated Cl- channel. Here, we report evidence for a lipid scrambling pathway along a groove harboring a lipid trail outside the ion permeation pore. This groove contains the binding pocket for niclosamide and 1PBC. Mutations of two residues in this groove specifically affect lipid scrambling. Whereas mutations of some residues in the binding pocket of niclosamide and 1PBC reduce their inhibition of TMEM16F-mediated Ca2+ influx and PS exposure, other mutations preferentially affect the ability of niclosamide and/or 1PBC to inhibit TMEM16F-mediated PS exposure, providing further support for separate pathways for ion permeation and lipid scrambling.

Identification of a conserved drug binding pocket in TMEM16 proteins.

The TMEM16 family of calcium-activated membrane proteins includes ten mammalian paralogs (TMEM16A-K) playing distinct physiological roles with some implicated in cancer and airway diseases. Their modulators with therapeutic potential include 1PBC, a potent inhibitor with anti-tumoral properties, and the FDA-approved drug niclosamide that targets TMEM16F to inhibit syncytia formation induced by SARS-CoV-2 infection. Here, we report cryo-EM structures of TMEM16F associated with 1PBC and niclosamide, revealing that both molecules bind the same drug binding pocket. We functionally and computationally validate this binding pocket in TMEM16A as well as TMEM16F, thereby showing that drug modulation also involves residues that are not conserved between TMEM16A and TMEM16F. This study establishes a much-needed structural framework for the development of more potent and more specific drug molecules targeting TMEM16 proteins.

A SWELL time to develop the molecular pharmacology of the volume-regulated anion channel (VRAC).

Newly emerging roles of LRRC8 volume-regulated anion channels (VRAC) raise important questions about the therapeutic potential of VRAC in the treatment of epilepsy, type 2 diabetes, and other human diseases. A critical barrier to evaluating whether VRAC represents a viable drug target is the lack of potent and specific small-molecule inhibitors and activators of the channel. Here we review recent progress in developing the molecular pharmacology of VRAC made by screening a library of FDA-approved drugs for novel channel modulators. We discuss the discovery and characterization of cysteinyl leukotriene receptor antagonists Pranlukast and Zafirlukast as novel VRAC inhibitors, and zinc pyrithione (ZPT), which apparently activates VRAC through a reactive oxygen species (ROS)-dependent mechanism. These ongoing efforts set the stage for developing a pharmacological toolkit for probing the integrative physiology, molecular pharmacology, and therapeutic potential of VRAC.

Huge subcutaneous extraperitoneal pseudocyst after migration of the ventriculoperitoneal shunt catheter.

The formation of an intraperitoneal pseudocyst as a complication of ventriculoperitoneal shunts is well known. However, the formation of a pseudocyst at the subcutaneous extraperitoneal abdominal space is unusual and likely secondary to the migration of the peritoneal catheter. We present a 53-year-old male who had placement of a ventriculoperitoneal shunt for hydrocephalus secondary to a vestibular schwannoma. Five months later, he presented with an enormously distended abdomen. Investigations showed the peritoneal catheter in the extraperitoneal space within a large right lower quadrant abdominal wall pseudocyst. The patient was taken to the operating theatre, and the shunt was externalised at the original abdominal incision. Approximately 3 L of cerebrospinal fluid were aspirated from the distal peritoneal catheter. After negative cultures, a new peritoneal catheter was placed intraperitoneally at the contralateral lower abdominal quadrant. The contralateral quadrant was utilised to prevent fluid accumulation into the old extraperitoneal cavity.

Zinc pyrithione activates the volume-regulated anion channel through an antioxidant-sensitive mechanism.

Leucine-rich repeat-containing 8 (LRRC8) volume-regulated anion channels (VRACs) play important physiological roles in diverse cell types and may represent therapeutic targets for various diseases. To date, however, the pharmacological tools for evaluating the druggability of VRACs have been limited to inhibitors, as no activators of the channel have been reported. We therefore performed a fluorescence-based high-throughput screening (HTS) of 1,184 Food and Drug Administration-approved drugs for compounds that increase VRAC activity. The most potent VRAC potentiator identified was zinc pyrithione (ZPT), which is used commercially as an antifouling agent and for treating dandruff and other skin disorders. In intracellular Yellow Fluorescent Protein YFP(F46L/H148Q/I152L)-quenching assays, ZPT potentiates the rate and extent of swelling-induced iodide influx dose dependently with a half-maximal effective concentration (EC50) of 5.7 µM. Whole cell voltage-clamp experiments revealed that coapplication of hypotonic solution and 30 µM ZPT to human embryonic kidney 293 or human colorectal carcinoma 116 cells increases the rate of swelling-induced VRAC activation by approximately 10-fold. ZPT potentiates swelling-induced VRAC currents after currents have reached a steady state and activates currents in the absence of cell swelling. Neither ZnCl2 nor free pyrithione activated VRAC; however, treating cells with a mixture of ZnCl2 and pyrithione led to robust channel activation. Finally, the effects of ZPT on VRAC were inhibited by reactive oxygen species (ROS) scavenger N-acetylcysteine (NAC) and NAD(P)H oxidase inhibitor diphenyleneiodonium chloride, suggesting the mechanism of action involves ROS generation. The discovery of ZPT as a potentiator/activator of VRAC demonstrates the utility of HTS for identifying small-molecule modulators of VRAC and adds to a growing repertoire of pharmacological tool compounds for probing the molecular physiology and regulation of this important channel.

Predicting spatial patterns of soil bacteria under current and future environmental conditions.

Soil bacteria are largely missing from future biodiversity assessments hindering comprehensive forecasts of ecosystem changes. Soil bacterial communities are expected to be more strongly driven by pH and less by other edaphic and climatic factors. Thus, alkalinisation or acidification along with climate change may influence soil bacteria, with subsequent influences for example on nutrient cycling and vegetation. Future forecasts of soil bacteria are therefore needed. We applied species distribution modelling (SDM) to quantify the roles of environmental factors in governing spatial abundance distribution of soil bacterial OTUs and to predict how future changes in these factors may change bacterial communities in a temperate mountain area. Models indicated that factors related to soil (especially pH), climate and/or topography explain and predict part of the abundance distribution of most OTUs. This supports the expectations that microorganisms have specific environmental requirements (i.e., niches/envelopes) and that they should accordingly respond to environmental changes. Our predictions indicate a stronger role of pH over other predictors (e.g. climate) in governing distributions of bacteria, yet the predicted future changes in bacteria communities are smaller than their current variation across space. The extent of bacterial community change predictions varies as a function of elevation, but in general, deviations from neutral soil pH are expected to decrease abundances and diversity of bacteria. Our findings highlight the need to account for edaphic changes, along with climate changes, in future forecasts of soil bacteria.

LRRC8A homohexameric channels poorly recapitulate VRAC regulation and pharmacology.

Swelling-activated volume-regulated anion channels (VRACs) are heteromeric channels comprising LRRC8A and at least one other LRRC8 paralog. Cryoelectron microscopy (cryo-EM) structures of nonnative LRRC8A and LRRC8D homohexamers have been described. We demonstrate here that LRRC8A homohexamers poorly recapitulate VRAC functional properties. Unlike VRACs, LRRC8A channels heterologously expressed in Lrr8c-/- HCT116 cells are poorly activated by low intracellular ionic strength (µ) and insensitive to cell swelling with normal µ. Combining low µ with swelling modestly activates LRRC8A, allowing characterization of pore properties. VRACs are strongly inhibited by 10 µM 4-[(2-butyl-6,7-dichloro-2-cyclopentyl-2,3-dihydro-1-oxo-1H-inden-5-yl)oxy]butanoic acid (DCPIB) in a voltage-independent manner. In contrast, DCPIB block of LRRC8A is weak and voltage sensitive. Cryo-EM structures indicate that DCPIB block is dependent on arginine 103. Consistent with this, LRRC8A R103F mutants are insensitive to DCPIB. However, an LRRC8 chimeric channel in which R103 is replaced by a leucine at the homologous position is inhibited ∼90% by 10 µM DCPIB in a voltage-independent manner. Coexpression of LRRC8A and LRRC8C gives rise to channels with DCPIB sensitivity that is strongly µ dependent. At normal intracellular µ, LRRC8A + LRRC8C heteromers exhibit strong, voltage-independent DCPIB block that is insensitive to R103F. DCPIB inhibition is greatly reduced and exhibits voltage dependence with low intracellular µ. The R103F mutation has no effect on maximal DCPIB inhibition but eliminates voltage dependence under low µ conditions. Our findings demonstrate that the LRRC8A cryo-EM structure and the use of heterologously expressed LRRC8 heteromeric channels pose significant limitations for VRAC mutagenesis-based structure-function analysis. Native VRAC function is most closely mimicked by chimeric LRRC8 homomeric channels.

Greater topoclimatic control of above- versus below-ground communities.

Assessing the degree to which climate explains the spatial distributions of different taxonomic and functional groups is essential for anticipating the effects of climate change on ecosystems. Most effort so far has focused on above-ground organisms, which offer only a partial view on the response of biodiversity to environmental gradients. Here including both above- and below-ground organisms, we quantified the degree of topoclimatic control on the occurrence patterns of >1,500 taxa and phylotypes along a c. 3,000 m elevation gradient, by fitting species distribution models. Higher model performances for animals and plants than for soil microbes (fungi, bacteria and protists) suggest that the direct influence of topoclimate is stronger on above-ground species than on below-ground microorganisms. Accordingly, direct climate change effects are predicted to be stronger for above-ground than for below-ground taxa, whereas factors expressing local soil microclimate and geochemistry are likely more important to explain and forecast the occurrence patterns of soil microbiota. Detailed mapping and future scenarios of soil microclimate and microhabitats, together with comparative studies of interacting and ecologically dependent above- and below-ground biota, are thus needed to understand and realistically forecast the future distribution of ecosystems.

The LRRC8 volume-regulated anion channel inhibitor, DCPIB, inhibits mitochondrial respiration independently of the channel.

There has been a resurgence of interest in the volume-regulated anion channel (VRAC) since the recent cloning of the LRRC8A-E gene family that encodes VRAC. The channel is a heteromer comprised of LRRC8A and at least one other family member; disruption of LRRC8A expression abolishes VRAC activity. The best-in-class VRAC inhibitor, DCPIB, suffers from off-target activity toward several different channels and transporters. Considering that some anion channel inhibitors also suppress mitochondrial respiration, we systematically explored whether DCPIB inhibits respiration in wild type (WT) and LRRC8A-knockout HAP-1 and HEK-293 cells. Knockout of LRRC8A had no apparent effects on cell morphology, proliferation rate, mitochondrial content, or expression of several mitochondrial genes in HAP-1 cells. Addition of 10 µM DCPIB, a concentration typically used to inhibit VRAC, suppressed basal and ATP-linked respiration in part through uncoupling the inner mitochondrial membrane (IMM) proton gradient and membrane potential. Additionally, DCPIB inhibits the activity of complex I, II, and III of the electron transport chain (ETC). Surprisingly, the effects of DCPIB on mitochondrial function are also observed in HAP-1 and HEK-293 cells which lack LRRC8A expression. Finally, we demonstrate that DCPIB activates ATP-inhibitable potassium channels comprised of heterologously expressed Kir6.2 and SUR1 subunits. These data indicate that DCPIB suppresses mitochondrial respiration and ATP production by dissipating the mitochondrial membrane potential and inhibiting complexes I-III of the ETC. They further justify the need for the development of sharper pharmacological tools for evaluating the integrative physiology and therapeutic potential of VRAC in human diseases.

CysLT1 receptor antagonists pranlukast and zafirlukast inhibit LRRC8-mediated volume regulated anion channels independently of the receptor.

Volume-regulated anion channels (VRACs) encoded by the leucine-rich repeat containing 8 (LRRC8) gene family play critical roles in myriad cellular processes and might represent druggable targets. The dearth of pharmacological compounds available for studying VRAC physiology led us to perform a high-throughput screen of 1,184 of US Food and Drug Administration-approved drugs for novel VRAC modulators. We discovered the cysteinyl leukotriene receptor 1 (CysLT1R) antagonist, pranlukast, as a novel inhibitor of endogenous VRAC expressed in human embryonic kidney 293 (HEK293) cells. Pranlukast inhibits VRAC voltage-independently, reversibly, and dose-dependently with a maximal efficacy of only ~50%. The CysLT1R pathway has been implicated in activation of VRAC in other cell types, prompting us to test whether pranlukast requires the CysLT1R for inhibition of VRAC. Quantitative PCR analysis demonstrated that CYSLTR1 mRNA is virtually undetectable in HEK293 cells. Furthermore, the CysLT1R agonist leukotriene D4 had no effect on VRAC activity and failed to stimulate Gq-coupled receptor signaling. Heterologous expression of the CysLT1R reconstituted LTD4-CysLT1R- Gq-calcium signaling in HEK293 cells but had no effect on VRAC inhibition by pranlukast. Finally, we show the CysLT1R antagonist zafirlukast inhibits VRAC with an IC50 of ~17 µM and does so with full efficacy. Our data suggest that both pranlukast and zafirlukast are likely direct channel inhibitors that work independently of the CysLT1R. This study provides clarifying insights into the putative role of leukotriene signaling in modulation of VRAC and identifies two new chemical scaffolds that can be used for development of more potent and specific VRAC inhibitors.

Archaeorhizomycetes Spatial Distribution in Soils Along Wide Elevational and Environmental Gradients Reveal Co-abundance Patterns With Other Fungal Saprobes and Potential Weathering Capacities.

Archaeorhizomycetes, a widespread fungal class with a dominant presence in many soil environments, contains cryptic filamentous species forming plant-root associations whose role in terrestrial ecosystems remains unclear. Here, we apply a correlative approach to identify the abiotic and biotic environmental variables shaping the distribution of this fungal group. We used a DNA sequencing dataset containing Archaeorhizomycetes sequences and environmental variables from 103 sites, obtained through a random-stratified sampling in the Western Swiss Alps along a wide elevation gradient (>2,500 m). We observed that the relative abundance of Archaeorhizomycetes follows a "humped-shaped" curve. Fitted linear and quadratic generalized linear models revealed that both climatic (minimum temperature, precipitation sum, growing degree-days) and edaphic (carbon, hydrogen, organic carbon, aluminum oxide, and phyllosilicates) factors contribute to explaining the variation in Archaeorhizomycetes abundance. Furthermore, a network inference topology described significant co-abundance patterns between Archaeorhizomycetes and other saprotrophic and ectomycorrhizal fungal taxa. Overall, our results provide strong support to the hypothesis that Archaeorhizomycetes in this area have clear ecological requirements along wide, elevation-driven abiotic and biotic gradients. Additionally, correlations to soil redox parameters, particularly with phyllosilicates minerals, suggest Archaeorhizomycetes might be implied in biological rock weathering. Such soil taxa-environment studies along wide gradients are thus a useful complement to latitudinal field observations and culture-based approaches to uncover the ecological roles of cryptic soil organisms.

Discovery, Characterization, and Effects on Renal Fluid and Electrolyte Excretion of the Kir4.1 Potassium Channel Pore Blocker, VU0134992.

The inward rectifier potassium (Kir) channel Kir4.1 (KCNJ10) carries out important physiologic roles in epithelial cells of the kidney, astrocytes in the central nervous system, and stria vascularis of the inner ear. Loss-of-function mutations in KCNJ10 lead to EAST/SeSAME syndrome, which is characterized by epilepsy, ataxia, renal salt wasting, and sensorineural deafness. Although genetic approaches have been indispensable for establishing the importance of Kir4.1 in the normal function of these tissues, the availability of pharmacological tools for acutely manipulating the activity of Kir4.1 in genetically normal animals has been lacking. We therefore carried out a high-throughput screen of 76,575 compounds from the Vanderbilt Institute of Chemical Biology library for small-molecule modulators of Kir4.1. The most potent inhibitor identified was 2-(2-bromo-4-isopropylphenoxy)-N-(2,2,6,6-tetramethylpiperidin-4-yl)acetamide (VU0134992). In whole-cell patch-clamp electrophysiology experiments, VU0134992 inhibits Kir4.1 with an IC50 value of 0.97 µM and is 9-fold selective for homomeric Kir4.1 over Kir4.1/5.1 concatemeric channels (IC50 = 9 µM) at -120 mV. In thallium (Tl+) flux assays, VU0134992 is greater than 30-fold selective for Kir4.1 over Kir1.1, Kir2.1, and Kir2.2; is weakly active toward Kir2.3, Kir6.2/SUR1, and Kir7.1; and is equally active toward Kir3.1/3.2, Kir3.1/3.4, and Kir4.2. This potency and selectivity profile is superior to Kir4.1 inhibitors amitriptyline, nortriptyline, and fluoxetine. Medicinal chemistry identified components of VU0134992 that are critical for inhibiting Kir4.1. Patch-clamp electrophysiology, molecular modeling, and site-directed mutagenesis identified pore-lining glutamate 158 and isoleucine 159 as critical residues for block of the channel. VU0134992 displayed a large free unbound fraction (fu) in rat plasma (fu = 0.213). Consistent with the known role of Kir4.1 in renal function, oral dosing of VU0134992 led to a dose-dependent diuresis, natriuresis, and kaliuresis in rats. Thus, VU0134992 represents the first in vivo active tool compound for probing the therapeutic potential of Kir4.1 as a novel diuretic target for the treatment of hypertension.

Meta-scale mountain grassland observatories uncover commonalities as well as specific interactions among plant and non-rhizosphere soil bacterial communities.

Interactions between plants and bacteria in the non-rhizosphere soil are rarely assessed, because they are less direct and easily masked by confounding environmental factors. By studying plant vegetation alliances and soil bacterial community co-patterning in grassland soils in 100 sites across a heterogeneous mountain landscape in the western Swiss Alps, we obtained sufficient statistical power to disentangle common co-occurrences and weaker specific interactions. Plant alliances and soil bacterial communities tended to be synchronized in community turnover across the landscape, largely driven by common underlying environmental factors, such as soil pH or elevation. Certain alliances occurring in distinct, local, environmental conditions were characterized by co-occurring specialist plant and bacterial species, such as the Nardus stricta and Thermogemmatisporaceae. In contrast, some generalist taxa, like Anthoxanthum odoratum and 19 Acidobacteria species, spanned across multiple vegetation alliances. Meta-scale analyses of soil bacterial community composition and vegetation surveys, complemented with local edaphic measurements, can thus prove useful to identify the various types of plant-bacteria interactions and the environments in which they occur.

Pore Polarity and Charge Determine Differential Block of Kir1.1 and Kir7.1 Potassium Channels by Small-Molecule Inhibitor VU590.

VU590 was the first publicly disclosed, submicromolar-affinity (IC50 = 0.2 µM), small-molecule inhibitor of the inward rectifier potassium (Kir) channel and diuretic target, Kir1.1. VU590 also inhibits Kir7.1 (IC50 ∼ 8 µM), and has been used to reveal new roles for Kir7.1 in regulation of myometrial contractility and melanocortin signaling. Here, we employed molecular modeling, mutagenesis, and patch clamp electrophysiology to elucidate the molecular mechanisms underlying VU590 inhibition of Kir1.1 and Kir7.1. Block of both channels is voltage- and K+-dependent, suggesting the VU590 binding site is located within the pore. Mutagenesis analysis in Kir1.1 revealed that asparagine 171 (N171) is the only pore-lining residue required for high-affinity block, and that substituting negatively charged residues (N171D, N171E) at this position dramatically weakens block. In contrast, substituting a negatively charged residue at the equivalent position in Kir7.1 enhances block by VU590, suggesting the VU590 binding mode is different. Interestingly, mutations of threonine 153 (T153) in Kir7.1 that reduce constrained polarity at this site (T153C, T153V, T153S) make wild-type and binding-site mutants (E149Q, A150S) more sensitive to block by VU590. The Kir7.1-T153C mutation enhances block by the structurally unrelated inhibitor VU714 but not by a higher-affinity analog ML418, suggesting that the polar side chain of T153 creates a barrier to low-affinity ligands that interact with E149 and A150. Reverse mutations in Kir1.1 suggest that this mechanism is conserved in other Kir channels. This study reveals a previously unappreciated role of membrane pore polarity in determination of Kir channel inhibitor pharmacology.

Local Environmental Factors Drive Divergent Grassland Soil Bacterial Communities in the Western Swiss Alps.

Mountain ecosystems are characterized by a diverse range of climatic and topographic conditions over short distances and are known to shelter a high biodiversity. Despite important progress, still little is known on bacterial diversity in mountain areas. Here, we investigated soil bacterial biogeography at more than 100 sampling sites randomly stratified across a 700-km2 area with 2,200-m elevation gradient in the western Swiss Alps. Bacterial grassland communities were highly diverse, with 12,741 total operational taxonomic units (OTUs) across 100 sites and an average of 2,918 OTUs per site. Bacterial community structure was correlated with local climatic, topographic, and soil physicochemical parameters with high statistical significance. We found pH (correlated with % CaO and % mineral carbon), hydrogen index (correlated with bulk gravimetric water content), and annual average number of frost days during the growing season to be among the groups of the most important environmental drivers of bacterial community structure. In contrast, bacterial community structure was only weakly stratified as a function of elevation. Contrasting patterns were discovered for individual bacterial taxa. Acidobacteria responded both positively and negatively to pH extremes. Various families within the Bacteroidetes responded to available phosphorus levels. Different verrucomicrobial groups responded to electrical conductivity, total organic carbon, water content, and mineral carbon contents. Alpine grassland bacterial communities are thus highly diverse, which is likely due to the large variety of different environmental conditions. These results shed new light on the biodiversity of mountain ecosystems, which were already identified as potentially fragile to anthropogenic influences and climate change. IMPORTANCE: This article addresses the question of how microbial communities in alpine regions are dependent on local climatic and soil physicochemical variables. We benefit from a unique 700-km2 study region in the western Swiss Alps region, which has been exhaustively studied for macro-organismal and fungal ecology, and for topoclimatic modeling of future ecological trends, but without taking into account soil bacterial diversity. Here, we present an in-depth biogeographical characterization of the bacterial community diversity in this alpine region across 100 randomly stratified sites, using 56 environmental variables. Our exhaustive sampling ensured the detection of ecological trends with high statistical robustness. Our data both confirm previously observed general trends and show many new detailed trends for a wide range of bacterial taxonomic groups and environmental parameters.

ML418: The First Selective, Sub-Micromolar Pore Blocker of Kir7.1 Potassium Channels.

The inward rectifier potassium (Kir) channel Kir7.1 (KCNJ13) has recently emerged as a key regulator of melanocortin signaling in the brain, electrolyte homeostasis in the eye, and uterine muscle contractility during pregnancy. The pharmacological tools available for exploring the physiology and therapeutic potential of Kir7.1 have been limited to relatively weak and nonselective small-molecule inhibitors. Here, we report the discovery in a fluorescence-based high-throughput screen of a novel Kir7.1 channel inhibitor, VU714. Site-directed mutagenesis of pore-lining amino acid residues identified glutamate 149 and alanine 150 as essential determinants of VU714 activity. Lead optimization with medicinal chemistry generated ML418, which exhibits sub-micromolar activity (IC50 = 310 nM) and superior selectivity over other Kir channels (at least 17-fold selective over Kir1.1, Kir2.1, Kir2.2, Kir2.3, Kir3.1/3.2, and Kir4.1) except for Kir6.2/SUR1 (equally potent). Evaluation in the EuroFins Lead Profiling panel of 64 GPCRs, ion-channels, and transporters for off-target activity of ML418 revealed a relatively clean ancillary pharmacology. While ML418 exhibited low CLHEP in human microsomes which could be modulated with lipophilicity adjustments, it showed high CLHEP in rat microsomes regardless of lipophilicity. A subsequent in vivo PK study of ML418 by intraperitoneal (IP) administration (30 mg/kg dosage) revealed a suitable PK profile (Cmax = 0.20 µM and Tmax = 3 h) and favorable CNS distribution (mouse brain/plasma Kp of 10.9 to support in vivo studies. ML418, which represents the current state-of-the-art in Kir7.1 inhibitors, should be useful for exploring the physiology of Kir7.1 in vitro and in vivo.

Plant species distributions along environmental gradients: do belowground interactions with fungi matter?

The distribution of plants along environmental gradients is constrained by abiotic and biotic factors. Cumulative evidence attests of the impact of biotic factors on plant distributions, but only few studies discuss the role of belowground communities. Soil fungi, in particular, are thought to play an important role in how plant species assemble locally into communities. We first review existing evidence, and then test the effect of the number of soil fungal operational taxonomic units (OTUs) on plant species distributions using a recently collected dataset of plant and metagenomic information on soil fungi in the Western Swiss Alps. Using species distribution models (SDMs), we investigated whether the distribution of individual plant species is correlated to the number of OTUs of two important soil fungal classes known to interact with plants: the Glomeromycetes, that are obligatory symbionts of plants, and the Agaricomycetes, that may be facultative plant symbionts, pathogens, or wood decayers. We show that including the fungal richness information in the models of plant species distributions improves predictive accuracy. Number of fungal OTUs is especially correlated to the distribution of high elevation plant species. We suggest that high elevation soil show greater variation in fungal assemblages that may in turn impact plant turnover among communities. We finally discuss how to move beyond correlative analyses, through the design of field experiments manipulating plant and fungal communities along environmental gradients.

DNA fingerprinting of pearls to determine their origins.

We report the first successful extraction of oyster DNA from a pearl and use it to identify the source oyster species for the three major pearl-producing oyster species Pinctada margaritifera, P. maxima and P. radiata. Both mitochondrial and nuclear gene fragments could be PCR-amplified and sequenced. A polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay in the internal transcribed spacer (ITS) region was developed and used to identify 18 pearls of unknown origin. A micro-drilling technique was developed to obtain small amounts of DNA while maintaining the commercial value of the pearls. This DNA fingerprinting method could be used to document the source of historic pearls and will provide more transparency for traders and consumers within the pearl industry.