Mummichog gill and operculum exhibit functionally consistent claudin-10 paralog profiles and Claudin-10c hypersaline response.

Claudin (Cldn)-10 tight junction (TJ) proteins are hypothesized to form the paracellular Na+ secretion pathway of hyposmoregulating mummichog (Fundulus heteroclitus) branchial epithelia. Organ-specific expression profiles showed that only branchial organs [the gill and opercular epithelium (OE)] exhibited abundant cldn-10 paralog transcripts, which typically increased following seawater (SW) to hypersaline (2SW) challenge. Post-translational properties, protein abundance, and ionocyte localization of Cldn-10c, were then examined in gill and OE. Western blot analysis revealed two Cldn-10c immunoreactive bands in the mummichog gill and OE at ∼29 kDa and ∼40 kDa. The heavier protein could be eliminated by glycosidase treatment, demonstrating the novel presence of a glycosylated Cldn-10c. Protein abundance of Cldn-10c increased in gill and OE of 2SW-exposed fish. Cldn-10c localized to the sides of gill and OE ionocyte apical crypts and partially colocalized with cystic fibrosis transmembrane conductance regulator and F-actin, consistent with TJ complex localization. Cldn-10c immunofluorescent intensity increased but localization was unaltered by 2SW conditions. In support of our hypothesis, cldn-10/Cldn-10 TJ protein dynamics in gill and OE of mummichogs and TJ localization are functionally consistent with the creation and maintenance of salinity-responsive, cation-selective pores that facilitate Na+ secretion in hyperosmotic environments.

Focal adhesion kinase and osmotic responses in ionocytes of Fundulus heteroclitus, a euryhaline teleost fish.

Cystic Fibrosis Transmembrane conductance Regulator (CFTR) anion channels are the regulated exit pathway in Cl- secretion by teleost salt secreting ionocytes of the gill and opercular epithelia of euryhaline teleosts. By confocal light immunocytochemistry using regular and phospho-antibodies directed against conserved sites, we found that killifish CFTR (kfCFTR) and the tyrosine kinase Focal Adhesion Kinase (FAK) phosphorylated at Y407 (FAKpY407) and FAKpY397 are colocalized at the apical membrane and in subjacent membrane vesicles of ionocytes. Hypotonic shock and the α-2 adrenergic agonist clonidine rapidly and reversibly inhibit Cl- secretion by isolated opercular epithelia, simultaneous with dephosphorylation of FAKpY407 and increased FAKpY397, located in the apical membrane of ionocytes in the opercular epithelium. FAKpY407 is re-phosphorylated at the apical membrane of ionocytes and Cl- secretion rapidly restored by hypertonic shock, detectable at 2 min., maximum at 5 min and still elevated at 30 min. In isolated opercular epithelia, the FAK phosphorylation inhibitor Y15 and p38MAP kinase inhibitor SB203580 significantly blunted the recovery of short-circuit current (Isc, equal to Cl- secretion rate) after hypertonic shock. The cSRC inhibitor saracatinib dephosphorylated FAKpY861 seen near tight junctions of pavement cells, and reduced the increase in epithelial resistance normally seen with clonidine inhibition of ion transport, while FAKpY397 was unaffected. The results show rapid osmosensitive responses in teleost fish ionocytes involve phosphorylation of CFTR by FAKpY407, an opposing role for FAKpY397 and a possible role for FAKpY861 in tight junction dynamics.

Biomimetic In Vitro Model of Cell Infiltration into Skin Scaffolds for Pre-Screening and Testing of Biomaterial-Based Therapies.

Due to great clinical need, research where different biomaterials are tested as 3D scaffolds for skin tissue engineering has increased. In vitro studies use a cell suspension that is simply pipetted onto the material and cultured until the cells migrate and proliferate within the 3D scaffold, which does not mimic the in vivo reality. Our aim was to engineer a novel biomimetic in vitro model that mimics the natural cell infiltration process occurring in wound healing, thus offering a realistic approach when pre-screening and testing new skin substitutes. Our model consists of porous membrane cell culture inserts coated with gelatin and seeded with human dermal fibroblasts, inside which two different commercially available dermal substitutes were placed. Several features relevant to the wound healing process (matrix contraction, cell infiltration and proliferation, integration of the biomaterial with the surrounding tissue, and secretion of exogenous cytokines and growth factors) were evaluated. Our results showed that cells spontaneously infiltrate the materials and that our engineered model is able to induce and detect subtle differences between different biomaterials. The model allows for room for improvements or "adds-on" and miniaturization and can contribute to the development of functional and efficient skin substitutes for burns and chronic wounds.

Sources of Collagen for Biomaterials in Skin Wound Healing.

Collagen is the most frequently used protein in the fields of biomaterials and regenerative medicine. Within the skin, collagen type I and III are the most abundant, while collagen type VII is associated with pathologies of the dermal-epidermal junction. The focus of this review is mainly collagens I and III, with a brief overview of collagen VII. Currently, the majority of collagen is extracted from animal sources; however, animal-derived collagen has a number of shortcomings, including immunogenicity, batch-to-batch variation, and pathogenic contamination. Recombinant collagen is a potential solution to the aforementioned issues, although production of correctly post-translationally modified recombinant human collagen has not yet been performed at industrial scale. This review provides an overview of current collagen sources, associated shortcomings, and potential resolutions. Recombinant expression systems are discussed, as well as the issues associated with each method of expression.

A MicroRNA-29 Mimic (Remlarsen) Represses Extracellular Matrix Expression and Fibroplasia in the Skin.

MicroRNA-29 (miR-29) negatively regulates fibrosis and is downregulated in multiple fibrotic organs and tissues, including in the skin. miR-29 mimics prevent pulmonary fibrosis in mouse models but have not previously been tested in the skin. This study aimed to identify pharmacodynamic biomarkers of miR-29 in mouse skin, to translate those biomarkers across multiple species, and to assess the pharmacodynamic activity of a miR-29b mimic (remlarsen) in a clinical trial. miR-29 biomarkers were selected based on gene function and mRNA expression using quantitative reverse transcriptase polymerase chain reaction. Those biomarkers comprised multiple collagens and other miR-29 direct and indirect targets and were conserved across species; remlarsen regulated their expression in mouse, rat, and rabbit skin wounds and in human skin fibroblasts in culture, while a miR-29 inhibitor reciprocally regulated their expression. Biomarker expression translated to clinical proof-of-mechanism; in a double-blinded, placebo-randomized, within-subject controlled clinical trial of single and multiple ascending doses of remlarsen in normal healthy volunteers, remlarsen repressed collagen expression and the development of fibroplasia in incisional skin wounds. These results suggest that remlarsen may be an effective therapeutic to prevent formation of a fibrotic scar (hypertrophic scar or keloid) or to prevent cutaneous fibrosis, such as scleroderma.

claudin-10 isoform expression and cation selectivity change with salinity in salt-secreting epithelia of Fundulusheteroclitus.

To provide insight into claudin (Cldn) tight junction (TJ) protein contributions to branchial salt secretion in marine teleost fishes, this study examined cldn-10 TJ protein isoforms of a euryhaline teleost (mummichog; Fundulus heteroclitus) in association with salinity change and measurements of transepithelial cation selectivity. Mummichogs were transferred from freshwater (FW) to seawater (SW, 35‰) and from SW to hypersaline SW (2SW, 60‰) in a time course with transfer control groups (FW to FW, and SW to SW). FW to SW transfer increased mRNA abundance of cldn-10d and cldn-10e twofold, whilst cldn-10c and cldn-10f transcripts were unchanged. Transfer from SW to 2SW did not alter cldn-10d, and transiently altered cldn-10e abundance, but increased cldn-10c and cldn-10f fourfold. This was coincident with an increased number of single-stranded junctions (observed by transmission electron microscopy). For both salinity transfers, (1) cldn-10e mRNA was acutely responsive (i.e. after 24 h), (2) other responsive cldn-10 isoforms increased later (3-7 days), and (3) cystic fibrosis transmembrane conductance regulator (cftr) mRNA was elevated in accordance with established changes in transcellular Cl- movement. Changes in mRNA encoding cldn-10c and -10f appeared linked, consistent with the tandem repeat locus in the Fundulus genome, whereas mRNA for tandem cldn-10d and cldn-10e seemed independent of each other. Cation selectivity sequence measured by voltage and conductance responses to artificial SW revealed Eisenman sequence VII: Na+>K+>Rb+∼Cs+>Li+ Collectively, these data support the idea that Cldn-10 TJ proteins create and maintain cation-selective pore junctions in salt-secreting tissues of teleost fishes.

Osmotic versus adrenergic control of ion transport by ionocytes of Fundulus heteroclitus in the cold.

In eurythermic vertebrates, acclimation to the cold may produce changes in physiological control systems. We hypothesize that relatively direct osmosensitive control will operate better than adrenergic receptor mediated control of ion transport in cold vs. warm conditions. Fish were acclimated to full strength seawater (SW) at 21°C and 5°C for four weeks, gill samples and blood were taken and opercular epithelia mounted in Ussing style chambers. Short-circuit current (Isc) at 21°C and 5°C (measured at acclimation temperature), was significantly inhibited by the α2-adrenergic agonist clonidine but the ED50 dose was significantly higher in cold conditions (93.8±16.4nM) than in warm epithelia (47.8±8.1nM) and the maximum inhibition was significantly lower in cold (-66.1±2.2%) vs. warm conditions (-85.6±1.3%), indicating lower sensitivity in the cold. ß-Adrenergic responses were unchanged. Hypotonic inhibition of Isc, was higher in warm acclimated (-95%), compared to cold acclimated fish (-75%), while hypertonic stimulations were the same, indicating equal responsiveness to hyperosmotic stimuli. Plasma osmolality was significantly elevated in cold acclimated fish and, by TEM, gill ionocytes from cold acclimated fish had significantly shorter mitochondria. These data are consistent with a shift in these eurythermic animals from complex adrenergic control to relatively simple biomechanical osmotic control of ion secretion in the cold.

Nitric oxide inhibition of NaCl secretion in the opercular epithelium of seawater-acclimated killifish, Fundulus heteroclitus.

Nitric oxide (NO) modulates epithelial ion transport pathways in mammals, but this remains largely unexamined in fish. We explored the involvement of NO in controlling NaCl secretion by the opercular epithelium of seawater killifish using an Ussing chamber approach. Pharmacological agents were used to explore the mechanism(s) triggering NO action. A modified Biotin-switch technique was used to investigate S-nitrosation of proteins. Stimulation of endogenous NO production via the nitric oxide synthase (NOS) substrate l-arginine (2.0 mmol l-1), and addition of exogenous NO via the NO donor SNAP (10-6 to 10-4 mol l-1), decreased the epithelial short-circuit current (Isc). Inhibition of endogenous NO production by the NOS inhibitor l-NAME (10-4 mol l-1) increased Isc and revealed a tonic control of ion transport by NO in unstimulated opercular epithelia. The NO scavenger PTIO (10-5 mol l-1) supressed the NO-mediated decrease in Isc, and confirmed that the effect observed was elicited by release of NO. The effect of SNAP on Isc was abolished by inhibitors of the soluble guanylyl cyclase (sGC), ODQ (10-6 mol l-1) and Methylene Blue (10-4 mol l-1), revealing NO signalling via the sGC/cGMP pathway. Incubation of opercular epithelium and gill tissues with SNAP (10-4 mol l-1) led to S-nitrosation of proteins, including Na+/K+-ATPase. Blocking of NOS with l-NAME (10-6 mol l-1) or scavenging of NO with PTIO during hypotonic shock suggested an involvement of NO in the hypotonic-mediated decrease in Isc Yohimbine (10-4 mol l-1), an inhibitor of α2-adrenoceptors, did not block NO effects, suggesting that NO is not involved in the α-adrenergic control of NaCl secretion.

Paracellular pathway remodeling enhances sodium secretion by teleost fish in hypersaline environments.

In vertebrate salt-secreting epithelia, Na(+) moves passively down an electrochemical gradient via a paracellular pathway. We assessed how this pathway is modified to allow Na(+) secretion in hypersaline environments. Mummichogs (Fundulus heteroclitus) acclimated to hypersaline [2× seawater (2SW), 64‰] for 30 days developed invasive projections of accessory cells with an increased area of tight junctions, detected by punctate distribution of CFTR (cystic fibrosis transmembrane conductance regulator) immunofluorescence and transmission electron miscroscopy of the opercular epithelia, which form a gill-like tissue rich in ionocytes. Distribution of CFTR was not explained by membrane raft organization, because chlorpromazine (50 µmol l(-1)) and filipin (1.5 µmol l(-1)) did not affect opercular epithelia electrophysiology. Isolated opercular epithelia bathed in SW on the mucosal side had a transepithelial potential (Vt) of +40.1±0.9 mV (N=24), sufficient for passive Na(+) secretion (Nernst equilibrium voltage≡ENa=+24.11 mV). Opercular epithelia from fish acclimated to 2SW and bathed in 2SW had higher Vt of +45.1±1.2 mV (N=24), sufficient for passive Na(+) secretion (ENa=+40.74 mV), but with diminished net driving force. Bumetanide block of Cl(-) secretion reduced Vt by 45% and 29% in SW and 2SW, respectively, a decrease in the driving force for Na(+) extrusion. Estimates of shunt conductance from epithelial conductance (Gt) versus short-circuit current (Isc) plots (extrapolation to zero Isc) suggested a reduction in total epithelial shunt conductance in 2SW-acclimated fish. In contrast, the morphological elaboration of tight junctions, leading to an increase in accessory-cell-ionocyte contact points, suggests an increase in local paracellular conductance, compensating for the diminished net driving force for Na(+) and allowing salt secretion, even in extreme salinities.

Cold acclimation allows regulation of chloride secretion in a eurythermic teleost fish Fundulus heteroclitus.

Fundulus heteroclitus (mummichog or common killifish) is an ideal model for ion transport regulation in chloride cells of the opercular epithelium (OE) and the response to thermal challenge. Mummichogs were acclimated to warm (20 °C) and cold (5 °C) seawater and opercular epithelia dissected and mounted in isolated Ussing-style epithelia chambers. The α2 adrenergic agonist clonidine inhibited the Cl(-) secretion (measured as short-circuit current, Isc), while the ß-adrenergic agonist isoproterenol and 1.0mM dibutyryl cyclic adenosine monophosphate (db-cAMP) plus 0.1mM isobutyl methylxanthine (IBMX) stimulated Isc in OE from warm and cold acclimated fish, measured at 20 °C. In contrast, rapid cooling partially inhibited Isc, but totally blocked the inhibition by clonidine and stimulation by isoproterenol and db-cAMP+IBMX in OE from warm-acclimated fish, while OE from cold-acclimated animals responded normally at 5 °C. Warming epithelia from 5 °C to 20 °C restored Isc and stimulation by db-cAMP+IBMX markedly increased Isc to levels similar to warm acclimated epithelia, while isoproterenol was much less effective. The isoproterenol insensitivity suggests a downregulation of ß-adrenergic receptors in the cold. We infer from present results and previous work (Buhariwalla et al. 2012) that cold shock of plasma membranes induces a phase shift from liquid to gel state that impaired plasma membrane protein mobility of necessary hormone regulatory functions, while cold acclimation preserved ion transport regulation via homeoviscous adaptation of plasma membrane lipids.

Cold acclimation of NaCl secretion in a eurythermic teleost: mitochondrial function and gill remodeling.

Active chloride secretion, measured as short-circuit current (Isc) in ionocytes of opercular epithelia (OE) in the eurythermic, euryoxic, and euryhaline killifish or mummichog (Fundulus heteroclitus) was studied in cold (5°C) and warm (20°C) acclimated fish to determine if homeoviscous adaptation aided chloride secretion in the cold. Isolated opercular epithelia were cooled from 30°C to 0.2°C for warm and cold acclimated fish; from 30 to 8°C, Isc decreased with Q10=1.68 for warm and Q10=1.56 for cold acclimated tissues. By Arrhenius plots, there is a critical temperature, 8°C, below which aerobic Isc decreased sharply (Q10=6.90 for warm and 4.23 for cold acclimated tissues), suggesting a shift in mitochondrial efficiency of oxidative phosphorylation. In anaerobic conditions (0.5mM NaCN; N2 saturation), chloride transport continued at a lower rate, and Isc decrease with cooling below 8°C was less pronounced (Q10=2.95 for warm and 3.08 for cold), suggesting a shift in transporter function in plasma membrane. Under anaerobic conditions, NaCl secretion at 20°C was reversibly inhibited by hypotonic shock, indicating normal regulation of transport. Chloride secretion in warm-acclimated fish was supported mostly (75% at 20°C) by aerobic metabolism, whereas that for cold-acclimated fish was lower (55% at 20°C), suggesting a greater reliance on anaerobic metabolism in the cold. Once acclimated to cold, ionocytes may be temporarily incapable of increasing their aerobic ATP supply, even when warmed to 30°C. In cold acclimated fish there was increased polyunsaturated fatty acid composition of gill epithelium (consistent with homeoviscous adaptation) and gill remodeling, wherein epithelial cells filled the interlamellar space (interlamellar cell mass, ILCM) by as much as 70%, thus increasing diffusion distance against passive ion gain. Most ionocytes in these thickened epithelial masses became taller, still connecting basal lamina with the environment, consistent with the continuing transport rates at low temperatures. Whereas the low aerobic scope of cold-acclimated fish and thickened gill epithelium is appropriate to winter inactivity, metabolic depression and anaerobiosis, the large aerobic scope of warm-acclimated fish favors active foraging at high temperatures.

The LCROSS cratering experiment.

As its detached upper-stage launch vehicle collided with the surface, instruments on the trailing Lunar Crater Observation and Sensing Satellite (LCROSS) Shepherding Spacecraft monitored the impact and ejecta. The faint impact flash in visible wavelengths and thermal signature imaged in the mid-infrared together indicate a low-density surface layer. The evolving spectra reveal not only OH within sunlit ejecta but also other volatile species. As the Shepherding Spacecraft approached the surface, it imaged a 25- to-30-meter-diameter crater and evidence of a high-angle ballistic ejecta plume still in the process of returning to the surface--an evolution attributed to the nature of the impactor.

The 50 year evolution of in vitro systems to reveal salt transport functions of teleost fish gills.

This short review traces the history of in vitro experimental methods that have been used to help elucidate the ion transport mechanisms of teleost fish gills. It begins with an isolated gill preparation published by Denis Bellamy in 1961 and progresses through many different approaches and concludes with current techniques. Among them are perfused gill arches, primary cultures of gill epithelia, isolated opercular skin preparations, whole embryos in vitro, the yolk-ball technique, dissociated gill epithelial cells, vibrating microprobe and scanning ion-selective microelectrodes; currently all are combined with molecular biological techniques. Each new approach brought new findings but is subject to certain limitations and each has contributed significantly to this important subfield of comparative physiology.

CFTR Cl- channel functional regulation by phosphorylation of focal adhesion kinase at tyrosine 407 in osmosensitive ion transporting mitochondria rich cells of euryhaline killifish.

Cystic fibrosis transmembrane conductance regulator (CFTR) anion channels are the regulated exit pathway in Cl(-) secretion by teleost mitochondria rich salt secreting (MR) cells of the gill and opercular epithelia of euryhaline teleosts. By confocal light immunocytochemistry, immunogold transmission electron microscopy (TEM), and co-immunoprecipitation, using regular and phospho-antibodies directed against conserved sites, we found that killifish CFTR (kfCFTR) and the tyrosine kinase focal adhesion kinase (FAK) phosphorylated at Y407 (FAK pY407) are colocalized in the apical membrane and in subjacent membrane vesicles of MR cells. We showed previously that basolateral FAK pY407, unlike other FAK phosphorylation sites, is osmosensitive and dephosphorylates during hypotonic shock of epithelial cells (Marshall et al., 2008). In the present study, we found that hypotonic shock and the alpha(2)-adrenergic agonist clonidine (neither of which affects cAMP levels) rapidly and reversibly inhibit Cl(-) secretion by isolated opercular membranes, simultaneous with dephosphorylation of FAK pY407, located in the apical membrane. FAK pY407 is rephosphorylated and Cl(-) secretion rapidly restored by hypertonic shock as well as by forskolin and isoproterenol, which operate via cAMP and protein kinase A. We conclude that hormone mediated, cAMP dependent and osmotically mediated, cAMP independent pathways converge on a mechanism to activate CFTR and Cl(-) secretion, possibly through tyrosine phosphorylation of CFTR by FAK.

Toward microRNA-based therapeutics for heart disease: the sense in antisense.

MicroRNAs act as negative regulators of gene expression by inhibiting the translation or promoting the degradation of target mRNAs. Because individual microRNAs often regulate the expression of multiple target genes with related functions, modulating the expression of a single microRNA can, in principle, influence an entire gene network and thereby modify complex disease phenotypes. Recent studies have identified signature expression patterns of microRNAs associated with pathological cardiac hypertrophy, heart failure, and myocardial infarction in humans and mouse models of heart disease. Gain- and loss-of-function studies in mice have revealed profound and unexpected functions for these microRNAs in numerous facets of cardiac biology, including the control of myocyte growth, contractility, fibrosis, and angiogenesis, providing glimpses of new regulatory mechanisms and potential therapeutic targets for heart disease. Especially intriguing is the discovery of a network of muscle-specific microRNAs embedded within myosin heavy chain genes, which control myosin expression and the response of the heart to stress and thyroid hormone signaling. Disease-inducing cardiac microRNAs can be persistently silenced in vivo through systemic delivery of antimiRs, allowing for the direct therapeutic modulation of disease mechanisms. Here, we summarize current knowledge of the roles of miRNAs in heart disease and consider the advantages and potential challenges of microRNA-based approaches compared to conventional drug-based therapies.

Distinct Na+/K+/2Cl- cotransporter localization in kidneys and gills of two euryhaline species, rainbow trout and killifish.

The kidney is an organ playing an important role in ion regulation in both freshwater (FW) and seawater (SW) fish. The mechanisms of ion regulation in the fish kidney are less well studied than that of their gills, especially at the level of transporter proteins. We have found striking differences in the pattern of Na(+)/K(+)/2Cl(-) cotransporter (NKCC) expression between species. In the killifish kidney, NKCC is apically localized in the distal and collecting tubules and basolaterally localized in the proximal tubules. However, in the SW killifish gill, NKCC is basolaterally co-localized with Na(+)/K(+)-ATPase, whereas in FW, NKCC immunoreactivity is primarily apical, although still colocalized within the same mitochondria-rich cell with basolateral Na(+)/K(+)-ATPase. Rainbow trout kidney has NKCC only in the apical membrane of the distal and collecting tubules in both environments, with no signal being detected in the proximal tubule. On the other hand, in the trout gill, NKCC is found basolaterally in both FW and SW environments. An important observation is that, in the gills of rainbow trout, the trailing edge of the filament possesses mostly Na(+)/K(+)-ATPase-positive but NKCC-negative mitochondria-rich cells, whereas in the region between and at the roots of the gill lamellae, most mitochondria-rich cells exhibit both Na(+)/K(+)-ATPase- and NKCC-positive immunoreactivity. These results suggest that the differential localization of transporters between the two species represents differences in function between these two euryhaline fishes with different life histories and strategies.

Dysregulation of microRNAs after myocardial infarction reveals a role of miR-29 in cardiac fibrosis.

Acute myocardial infarction (MI) due to coronary artery occlusion is accompanied by a pathological remodeling response that includes hypertrophic cardiac growth and fibrosis, which impair cardiac contractility. Previously, we showed that cardiac hypertrophy and heart failure are accompanied by characteristic changes in the expression of a collection of specific microRNAs (miRNAs), which act as negative regulators of gene expression. Here, we show that MI in mice and humans also results in the dysregulation of specific miRNAs, which are similar to but distinct from those involved in hypertrophy and heart failure. Among the MI-regulated miRNAs are members of the miR-29 family, which are down-regulated in the region of the heart adjacent to the infarct. The miR-29 family targets a cadre of mRNAs that encode proteins involved in fibrosis, including multiple collagens, fibrillins, and elastin. Thus, down-regulation of miR-29 would be predicted to derepress the expression of these mRNAs and enhance the fibrotic response. Indeed, down-regulation of miR-29 with anti-miRs in vitro and in vivo induces the expression of collagens, whereas over-expression of miR-29 in fibroblasts reduces collagen expression. We conclude that miR-29 acts as a regulator of cardiac fibrosis and represents a potential therapeutic target for tissue fibrosis in general.

Fundulus as the premier teleost model in environmental biology: opportunities for new insights using genomics.

A strong foundation of basic and applied research documents that the estuarine fish Fundulus heteroclitus and related species are unique laboratory and field models for understanding how individuals and populations interact with their environment. In this paper we summarize an extensive body of work examining the adaptive responses of Fundulus species to environmental conditions, and describe how this research has contributed importantly to our understanding of physiology, gene regulation, toxicology, and ecological and evolutionary genetics of teleosts and other vertebrates. These explorations have reached a critical juncture at which advancement is hindered by the lack of genomic resources for these species. We suggest that a more complete genomics toolbox for F. heteroclitus and related species will permit researchers to exploit the power of this model organism to rapidly advance our understanding of fundamental biological and pathological mechanisms among vertebrates, as well as ecological strategies and evolutionary processes common to all living organisms.

Double-stranded regions are essential design components of potent inhibitors of RISC function.

While microRNAs (miRNAs) are recognized as playing a critical role in regulating eukaryotic gene expression, both the mechanism by which these small, noncoding RNAs function and the genes they target remain elusive. Previous studies have shown that short, single-stranded 2'-O-methyl-modified oligonucleotides that are complementary to mature microRNA sequences can interact with the miRNA-RISC nucleoprotein complex and weakly inhibit miRNA function. Here we report the identification of secondary structural elements that enhance the potency of these molecules. Incorporation of highly structured, double-stranded flanking regions around the reverse complement core significantly increases inhibitor function and allows for multi-miRNA inhibition at subnanomolar concentrations. The improved functionality of these double-stranded miRNA inhibitors may provide insights into the miRNA mechanism by suggesting the possible importance of such structures in or near endogenous miRNA target sites.