Leucine-rich repeats and calponin homology containing 4 (Lrch4) regulates the innate immune response.

Toll-like receptors (TLRs) are pathogen-recognition receptors that trigger the innate immune response. Recent reports have identified accessory proteins that provide essential support to TLR function through ligand delivery and receptor trafficking. Herein, we introduce leucine-rich repeats (LRRs) and calponin homology containing 4 (Lrch4) as a novel TLR accessory protein. Lrch4 is a membrane protein with nine LRRs in its predicted ectodomain. It is widely expressed across murine tissues and has two expression variants that are both regulated by lipopolysaccharide (LPS). Predictive modeling indicates that Lrch4 LRRs conform to the horseshoe-shaped structure typical of LRRs in pathogen-recognition receptors and that the best structural match in the protein database is to the variable lymphocyte receptor of the jawless vertebrate hagfish. Silencing Lrch4 attenuates cytokine induction by LPS and multiple other TLR ligands and dampens the in vivo innate immune response. Lrch4 promotes proper docking of LPS in lipid raft membrane microdomains. We provide evidence that this is through regulation of lipid rafts as Lrch4 silencing reduces cell surface gangliosides, a metric of raft abundance, as well as expression and surface display of CD14, a raft-resident LPS co-receptor. Taken together, we identify Lrch4 as a broad-spanning regulator of the innate immune response and a potential molecular target in inflammatory disease.

Chlorophyll absorption and phytoplankton size information inferred from hyperspectral particulate beam attenuation.

Electromagnetic theory predicts spectral dependencies in extinction efficiency near a narrow absorption band for a particle with an index of refraction close to that of the medium in which it is immersed. These absorption band effects are anticipated in oceanographic beam-attenuation (beam-c) spectra, primarily due to the narrow red peak in absorption produced by the phytoplankton photopigment, chlorophyll a (Chl a). Here we present a method to obtain Chl a absorption and size information by analyzing an eigendecomposition of hyperspectral beam-c residuals measured in marine surface waters by an automatic underway system. We find that three principal modes capture more than 99% of the variance in beam-c residuals at wavelengths near the Chl a red absorption peak. The spectral shapes of the eigenvectors resemble extinction efficiency residuals attributed to the absorption band effects. Projection of the eigenvectors onto the beam-c residuals produces a time series of amplitude functions with absolute values that are strongly correlated to concurrent Chl a absorption line height (aLH) measurements (r values of 0.59 to 0.83) and hence provide a method to estimate Chl a absorption. Multiple linear regression of aLH on the amplitude functions enables an independent estimate of aLH, with RMSE of 3.19⋅10-3m-1 (3.3%) or log10-RMSE of 18.6%, and a raw-scale R2 value of 0.90 based on the Tara Oceans Expedition data. Relationships between the amplitude functions and the beam-c exponential slopes are in agreement with theory relating beam-c to the particle size distribution. Compared to multispectral analysis of beam-c slope, hyperspectral analysis of absorption band effects is anticipated to be relatively insensitive to the addition of nonpigmented particles and to monodispersion.

Effects of Ice Massage Prior to an Iontophoresis Treatment Using Dexamethasone Sodium Phosphate.

CONTEXT: Low current intensity iontophoresis treatments have increased skin perfusion over 700% from baseline potentially altering drug clearance from or diffusion to the targeted area. OBJECTIVE: To determine the effects of a preceding 10-minute ice massage on subcutaneous dexamethasone sodium phosphate (Dex-P) concentration and skin perfusion during and after a 4-mA iontophoresis treatment. DESIGN: Controlled laboratory study. SETTING: Research laboratory. PATIENTS OR OTHER PARTICIPANTS: Twenty-four participants (male = 12, female = 12; age = 25.6 [4.5] y, height = 173.9 [8.51] cm, mass = 76.11 [16.84] kg). INTERVENTION(S): Participants were randomly assigned into 2 groups: (1) pretreatment 10-minute ice massage and (2) no pretreatment ice massage. Treatment consisted of an 80-mA·minute (4 mA, 20 min) Dex-P iontophoresis treatment. Microdialysis probes (3 mm deep in the forearm) were used to assess Dex-P, dexamethasone (Dex), and its metabolite (Dex-Met) concentrations. Skin perfusion was measured using laser Doppler flowmetry. MAIN OUTCOME MEASURE(S): Microdialysis samples were collected at baseline, at conclusion of treatment, and every 20 minutes posttreatment for 60 minutes. Samples were analyzed to determine Dex-Total (Dex-Total = Dex-P + Dex + Dex-Met). Skin perfusion was calculated as a percentage change from baseline. A mixed-design analysis of variance was used to determine Dex-Total and skin perfusion difference between groups overtime. RESULTS: There was no difference between groups (P = .476), but [Dex-Total] significantly increased over the course of the iontophoresis and posttreatment time (P < .001). Dex-P was measured in 18 of 24 participants with a mean concentration of 0.67 (1.09) µg/mL. Skin perfusion was significantly greater in the no ice treatment group (P = .002). Peak skin perfusion reached 27.74% (47.49%) and 117.39% (103.45%) from baseline for the ice and no ice groups, respectively. CONCLUSIONS: Ice massage prior to iontophoresis does not alter the tissue [Dex-Total] even with less skin perfusion.

Divalent ion competition reveals reorganization of an RNA ion atmosphere upon folding.

Although RNA interactions with K+ and Mg2+ have been studied extensively, much less is known about the third most abundant cation in bacterial cells, putrescine2+, and how RNA folding might be influenced by the three ions in combination. In a new approach, we have observed the competition between Mg2+ and putrescine2+ (in a background of K+) with native, partially unfolded and highly extended conformations of an adenine riboswitch aptamer. With the native state, putrescine2+ is a weak competitor when the ratio of the excess Mg2+ (which neutralizes phosphate charge) to RNA is very low, but becomes much more effective at replacing Mg2+ as the excess Mg2+ in the RNA ion atmosphere increases. Putrescine2+ is even more effective in competing Mg2+ from the extended conformation, independent of the Mg2+ excess. To account for these and other results, we propose that both ions closely approach the surface of RNA secondary structure, but the completely folded RNA tertiary structure develops small pockets of very negative electrostatic potential that are more accessible to the compact charge of Mg2+. The sensitivity of RNA folding to the combination of Mg2+ and putrescine2+ found in vivo depends on the architectures of both the unfolded and native conformations.

Effects of Long Duration Low Intensity Ultrasound for Active Trapezius Trigger Points: A Randomized Clinical Trial.

CONTEXT: Upper trapezius trigger points can be debilitating and often lead to chronic pain. OBJECTIVE: To determine if long duration low intensity therapeutic ultrasound is effective at treating upper trapezius trigger point pain compared to an inactive placebo treatment. DESIGN: Randomized control trial. SETTING: Research laboratory. PATIENTS OR OTHER PARTICIPANTS: Fifty-one participants (19 males, 32 females, age = 31.8 ± 12.5 y, ht = 168.6 ± 15.9 cm, mass = 77.1 ± 16.1 kg) diagnosed with chronic upper trapezius pain. INTERVENTIONS: Participants were randomly assigned into an active (n = 32) or sham (n = 19) treatment group. Treatments were applied at least 3 times a week for 4 weeks and each ultrasound treatment lasted 4 h. MAIN OUTCOME MEASURES: Participants completed a daily numeric rating scale (NRS) pain diary. Pain pressure threshold was assessed via a pressure algometer. Repeated-measures ANCOVAs were used to examine differences over time and between groups. RESULTS: Pain on the NRS was significantly reduced from baseline (5.4 ± 1.7) to the conclusion of the study (4.2 ± 1.6) (F4,254 = 7.747, P < 0.001); however, the reduction did not significantly differ between the two groups (F4,254 = 1.096, P = 0.359). There was an average reduction in pain of 1.1 ± 1.8 points over the course of a 4 h treatment (F4,254 = 18.67, P = 0.001), but this reduction did not differ between groups, (F4,254 = 0.701, P = 0.592). Pain pressure threshold improved by 0.46 ± 1.29 and 0.44 ± 1.73 kg for active and sham groups, respectively, but was not different between groups (F2,142 = 1.168, P = 0.314). CONCLUSIONS: Four-weeks of low intensity long duration ultrasound reduced upper trapezius trigger point pain, but the improvement is attributed to a placebo effect.

Computational evaluation of exome sequence data using human and model organism phenotypes improves diagnostic efficiency.

PURPOSE: Medical diagnosis and molecular or biochemical confirmation typically rely on the knowledge of the clinician. Although this is very difficult in extremely rare diseases, we hypothesized that the recording of patient phenotypes in Human Phenotype Ontology (HPO) terms and computationally ranking putative disease-associated sequence variants improves diagnosis, particularly for patients with atypical clinical profiles. METHODS: Using simulated exomes and the National Institutes of Health Undiagnosed Diseases Program (UDP) patient cohort and associated exome sequence, we tested our hypothesis using Exomiser. Exomiser ranks candidate variants based on patient phenotype similarity to (i) known disease-gene phenotypes, (ii) model organism phenotypes of candidate orthologs, and (iii) phenotypes of protein-protein association neighbors. RESULTS: Benchmarking showed Exomiser ranked the causal variant as the top hit in 97% of known disease-gene associations and ranked the correct seeded variant in up to 87% when detectable disease-gene associations were unavailable. Using UDP data, Exomiser ranked the causative variant(s) within the top 10 variants for 11 previously diagnosed variants and achieved a diagnosis for 4 of 23 cases undiagnosed by clinical evaluation. CONCLUSION: Structured phenotyping of patients and computational analysis are effective adjuncts for diagnosing patients with genetic disorders.Genet Med 18 6, 608-617.

APOε4 is associated with enhanced in vivo innate immune responses in human subjects.

BACKGROUND: The genetic determinants of the human innate immune response are poorly understood. Apolipoprotein (Apo) E, a lipid-trafficking protein that affects inflammation, has well-described wild-type (ε3) and disease-associated (ε2 and ε4) alleles, but its connection to human innate immunity is undefined. OBJECTIVE: We sought to define the relationship of APOε4 to the human innate immune response. METHODS: We evaluated APOε4 in several functional models of the human innate immune response, including intravenous LPS challenge in human subjects, and assessed APOε4 association to organ injury in patients with severe sepsis, a disease driven by dysregulated innate immunity. RESULTS: Whole blood from healthy APOε3/APOε4 volunteers induced higher cytokine levels on ex vivo stimulation with Toll-like receptor (TLR) 2, TLR4, or TLR5 ligands than blood from APOε3/APOε3 patients, whereas TLR7/8 responses were similar. This was associated with increased lipid rafts in APOε3/APOε4 monocytes. By contrast, APOε3/APOε3 and APOε3/APOε4 serum neutralized LPS equivalently and supported similar LPS responses in Apoe-deficient macrophages, arguing against a differential role for secretory APOE4 protein. After intravenous LPS, APOε3/APOε4 patients had higher hyperthermia and plasma TNF-α levels and earlier plasma IL-6 than APOε3/APOε3 patients. APOE4-targeted replacement mice displayed enhanced hypothermia, plasma cytokines, and hepatic injury and altered splenic lymphocyte apoptosis after systemic LPS compared with APOE3 counterparts. In a cohort of 828 patients with severe sepsis, APOε4 was associated with increased coagulation system failure among European American patients. CONCLUSIONS: APOε4 is a determinant of the human innate immune response to multiple TLR ligands and associates with altered patterns of organ injury in human sepsis.

Multiple hybridization events, polyploidy and low postmating isolation entangle the evolution of neotropical species of Epidendrum (Orchidaceae).

BACKGROUND: Hybridization and polyploidy are central processes in evolution and speciation. These mechanisms often lead to complex patterns of genetic variation and the creation of novel genotypes, which may establish if they become isolated from gene flow. However, in the absence of reproductive isolation, species boundaries might easily be disrupted. Here, we used a combination of AFLPs, chloroplast DNA markers and flow cytometry to investigate the evolutionary outcomes of hybridization between two endemic Ecuadorian species of Epidendrum (E. madsenii and E. rhopalostele) in three hybrid zones. Postmating isolation was also quantified to determine the role of this barrier in restraining gene flow between hybrids and the parental species. In addition, future ecological niche models were constructed to predict the outcomes of hybridization between these species. RESULTS: Our results confirmed the presence of hybrids in all hybrid zones, but revealed that a third parental species (E. falcisepalum) has contributed to one of the hybrid zones studied. Backcross genotypes were frequent in all hybrid zones, which was in accordance with the absence of strong reproductive barriers. The process of hybridization was highly asymmetric and followed in some cases by polyploidy. The projection of future niche models predicted a severe reduction in the area suitable for the occurrence of these species, although favorable conditions will still occur for the existence of the current hybrid zones. CONCLUSIONS: The recurrent process of hybridization has compromised the genetic integrity of the parental species. Most individuals of the parental species can no longer be considered as pure-bred individuals because most were classified as backcrossed hybrids. Novel genetic lineages occur in all hybrid zones implying that hybrids are fertile and can compete with the parental species. These results, together with the prediction of suitable conditions for the future occurrence of these hybrid zones, highlight the importance of conserving these geographic areas as sources of novel taxonomic entities.

Rock outcrop orchids reveal the genetic connectivity and diversity of inselbergs of northeastern Brazil.

BACKGROUND: Because of their fragmented nature, inselberg species are interesting biological models for studying the genetic consequences of disjoint populations. Inselbergs are commonly compared with oceanic islands, as most of them display a marked ecological isolation from the surrounding area. The isolation of these rock outcrops is reflected in the high number of recorded endemic species and the strong floristic differences between individual inselbergs and adjacent habitats. We examined the genetic connectivity of orchids Epidendrum cinnabarinum and E. secundum adapted to Neotropical inselbergs of northeastern Brazil. Our goals were to identify major genetic divergences or disjunctions across the range of the species and to investigate potential demographic and evolutionary mechanisms leading to lineage divergence in Neotropical mountain ecosystems. RESULTS: Based on plastid markers, high genetic differentiation was found for E. cinnabarinum (FST = 0.644) and E. secundum (FST = 0.636). Haplotypes were not geographically structured in either taxon, suggesting that restricted gene flow and genetic drift may be significant factors influencing the diversification of these inselberg populations. Moreover, strong differentiation was found between populations over short spatial scales, indicating substantial periods of isolation among populations. For E. secundum, nuclear markers indicated higher gene flow by pollen than by seeds. CONCLUSIONS: The comparative approach adopted in this study contributed to the elucidation of patterns in both species. Our results confirm the ancient and highly isolated nature of inselberg populations. Both species showed similar patterns of genetic diversity and structure, highlighting the importance of seed-restricted gene flow and genetic drift as drivers of plant diversification in terrestrial islands such as inselbergs.

ATP binding cassette transporter G1 deletion induces IL-17-dependent dysregulation of pulmonary adaptive immunity.

Mice with genetic deletion of the cholesterol transporter ATP binding cassette G1 (ABCG1) have pulmonary lipidosis and enhanced innate immune responses in the airway. Whether ABCG1 regulates adaptive immune responses to the environment is unknown. To this end, Abcg1(+/+) and Abcg1(-/-) mice were sensitized to OVA via the airway using low-dose LPS as an adjuvant, and then challenged with OVA aerosol. Naive Abcg1(-/-) mice displayed increased B cells, CD4(+) T cells, CD8(+) T cells, and dendritic cells (DCs) in lung and lung-draining mediastinal lymph nodes, with lung CD11b(+) DCs displaying increased CD80 and CD86. Upon allergen sensitization and challenge, the Abcg1(-/-) airway, compared with Abcg1(+/+), displayed reduced Th2 responses (IL-4, IL-5, eosinophils), increased neutrophils and IL-17, but equivalent airway hyperresponsiveness. Reduced Th2 responses were also found using standard i.p. OVA sensitization with aluminum hydroxide adjuvant. Mediastinal lymph nodes from airway-sensitized Abcg1(-/-) mice produced reduced IL-5 upon ex vivo OVA challenge. Abcg1(-/-) CD4(+) T cells displayed normal ex vivo differentiation, whereas Abcg1(-/-) DCs were found paradoxically to promote Th2 polarization. Th17 cells, IL-17(+) γδT cells, and IL-17(+) neutrophils were all increased in Abcg1(-/-) lungs, suggesting Th17 and non-Th17 sources of IL-17 excess. Neutralization of IL-17 prior to challenge normalized eosinophils and reduced neutrophilia in the Abcg1(-/-) airway. We conclude that Abcg1(-/-) mice display IL-17-mediated suppression of eosinophilia and enhancement of neutrophilia in the airway following allergen sensitization and challenge. These findings identify ABCG1 as a novel integrator of cholesterol homeostasis and adaptive immune programs.

Comparison of interactions of diamine and Mg²âº with RNA tertiary structures: similar versus differential effects on the stabilities of diverse RNA folds.

Cations play a large role in stabilizing the native state of RNA in vivo. In addition to Mg²âº, putrescine²âº is an abundant divalent cation in bacterial cells, but its effect on the folding of RNA tertiary structure has not been widely explored. In this study, we look at how the stabilities of four structured RNAs, each with a different degree of dependence on K⁺ and Mg²âº, are affected by putrescine²âº relative to Mg²âº. Through the use of thermal melts, we observe that (i) at a given concentration, putrescine²âº is less effective than Mg²âº at stabilizing RNA, (ii) the stability imparted to RNA by various diamines is a function of charge density (average separation distance between charges) as well as the flexibility of the counterion, and (iii) when Mg²âº is already present in a buffer, further addition of putrescine²âº may either destabilize or stabilize RNA structure, depending on whether the native RNA does or does not chelate Mg²âº ion, respectively. At ion concentrations likely to be found in vivo, the effect of putrescine²âº on the free energy of folding of an RNA tertiary structure is probably quite small compared to that of Mg²âº, but the ability of mixed Mg²âº/putrescine²âº environments to (in effect) discriminate between different RNA architectures suggests that, in some cells, the evolution of functional RNA structures may have been influenced by the presence of putrescine²âº.

Variations in definitions of mortality have little influence on neonatal intensive care unit performance ratings.

OBJECTIVE: To measure the influence of varying mortality time frames on performance rankings among regional neonatal intensive care units (NICUs) in a large state. STUDY DESIGN: We performed a cross-sectional data analysis of very low birth weight infants receiving care at 24 level 3 NICUs. We tested the effect of 4 definitions of mortality: (1) death between admission and end of birth hospitalization or up to 366 days; (2) death between 12 hours of age and the end of birth hospitalization or up to 366 days; (3) death between admission and 28 days; and (4) death between 12 hours of age and 28 days. NICUs were ranked by quantifying their deviation from risk-adjusted expected mortality and dividing them into 3 tiers: top 6, bottom 6, and in between. RESULTS: There was wide interinstitutional variation in risk-adjusted mortality for each definition (observed minus expected z-score range, -6.08 to 3.75). However, mortality-based NICU rankings and classification into performance tiers were very similar for all institutions in each of our time frames. Among all 4 definitions, NICU rank correlations were high (>0.91). Few NICUs changed relative to a neighboring tier with changes in definitions, and none changed by more than one tier. CONCLUSION: The time frame used to ascertain mortality had little effect on comparative NICU performance.

Risk-adjusting hospital mortality using a comprehensive electronic record in an integrated health care delivery system.

OBJECTIVE: Using a comprehensive inpatient electronic medical record, we sought to develop a risk-adjustment methodology applicable to all hospitalized patients. Further, we assessed the impact of specific data elements on model discrimination, explanatory power, calibration, integrated discrimination improvement, net reclassification improvement, performance across different hospital units, and hospital rankings. DESIGN: Retrospective cohort study using logistic regression with split validation. PARTICIPANTS: A total of 248,383 patients who experienced 391,584 hospitalizations between January 1, 2008 and August 31, 2011. SETTING: Twenty-one hospitals in an integrated health care delivery system in Northern California. RESULTS: Inpatient and 30-day mortality rates were 3.02% and 5.09%, respectively. In the validation dataset, the greatest improvement in discrimination (increase in c statistic) occurred with the introduction of laboratory data; however, subsequent addition of vital signs and end-of-life care directive data had significant effects on integrated discrimination improvement, net reclassification improvement, and hospital rankings. Use of longitudinally captured comorbidities did not improve model performance when compared with present-on-admission coding. Our final model for inpatient mortality, which included laboratory test results, vital signs, and care directives, had a c statistic of 0.883 and a pseudo-R of 0.295. Results for inpatient and 30-day mortality were virtually identical. CONCLUSIONS: Risk-adjustment of hospital mortality using comprehensive electronic medical records is feasible and permits one to develop statistical models that better reflect actual clinician experience. In addition, such models can be used to assess hospital performance across specific subpopulations, including patients admitted to intensive care.

Folding of RNA tertiary structure: Linkages between backbone phosphates, ions, and water.

The functional forms of many RNAs have compact architectures. The placement of phosphates within such structures must be influenced not only by the strong electrostatic repulsion between phosphates, but also by networks of interactions between phosphates, water, and mobile ions. This review first explores what has been learned of the basic thermodynamic constraints on these arrangements from studies of hydration and ions in simple DNA molecules, and then gives an overview of what is known about ion and water interactions with RNA structures. A brief survey of RNA crystal structures identifies several interesting architectures in which closely spaced phosphates share hydration shells or phosphates are buried in environments that provide intramolecular hydrogen bonds or site-bound cations. Formation of these structures must require strong coupling between the uptake of ions and release of water.

Successful short-term and long-term treatment of melasma and postinflammatory hyperpigmentation using vitamin C with a full-face iontophoresis mask and a mandelic/malic acid skin care regimen.

BACKGROUND: Treatment of melasma and postinflammatory hyperpigmentation is often challenging. No ideal short-term and long-term treatment is available. Vitamin C alone and in combination with iontophoresis has been studied and found to be useful; however, no long-term studies have been published. METHODS: In this study, 35 patients (34 female, 1 male) were treated with a novel full-face iontophoresis mask (FFIM) and a proprietary vitamin C (ascorbyl glucoside) preparation. Patients received one in-office treatment and 12 to 24 at-home treatments over 1 to 2 months in conjunction with a strict maintenance regimen consisting of a mandelic/malic acid skin care regimen, broad-spectrum ultraviolet A/ultraviolet B sunblock, a wide-brimmed hat, and sun-avoidance behavior. Follow-up after the initial in-office treatment ranged from 1 to 54 months (mean, 26 months). Four independent observers graded improvement of melasma and PIH using a 4-point scale. Before the study, high-performance liquid chromatography was used to verify iontophoretic penetration of vitamin C into the skin to a level of 0.2 cm in healthy volunteers (2 male, 2 female). RESULTS: A mean 73% improvement in abnormal pigmentation was observed at the end of FFIM/vitamin C treatment. Greater than 25% improvement was observed in 32 of 35 patients, and greater than 50% improvement in 22 of 35 patients. Melasma Area and Severity Index scores demonstrated substantial improvement from baseline for all patients, with a mean improvement of 15.7. CONCLUSIONS: Full-face iontophoresis of vitamin C appears to be an effective short-term treatment for melasma and postinflammatory hyperpigmentation. A protocol of strict sun avoidance in combination with a mandelic/malic acid skin care regimen appears to be useful in maintaining the improvement.

Contributions of muscle fatigue to a neuromuscular neck injury in female ballroom dancers.

OBJECTIVE: To investigate the etiology of a loss-of-control neck injury in international modern (IM) dancesport dancers. A comparison to identify if dancers with neck injury have a greater decrease in median frequency in electromyography (EMG) than non-injured dancers. SUBJECTS: Twenty female subjects (mean age 21.6 ± 3.0 yrs, height 167.1 ± 4.3 cm, weight 59.1 ± 5.2 kg, mean BMI 21.1 ± 1.2) with minimum 1-year experience in competitive IM dancesport. measurements: EMG activity from the left upper trapezius, left splenius capitius, and right sternocleidomastoid muscles before and after dancing the five IM dances. Extension, lateral flexion, and neck length were also measured. RESULTS: There was no significant difference in all measurements. CONCLUSIONS: Although we did not find the etiology of this neck injury, this was the first research into this injury. Future research could study the different IM dances, compare different competition levels, or the amount of force placed on the neck when dancing.

Genetic diversity and structure in two epiphytic orchids from the montane forests of southern Ecuador: The role of overcollection on Masdevallia rosea in comparison with the widespread Pleurothallis lilijae.

Ecuador has a high diversity of orchids, but little is known about levels of genetic diversity for the great majority of species. Understanding how orchids might adapt to changes is crucial as deforestation and fragmentation of forest ecosystems threaten the survival of many epiphytic orchids that depend on other species, such as fungi and their host trees, for germination, growth, and establishment. Overcollection and the illegal trade are also major concerns for the survival of wild populations of orchids. Despite increasing awareness, effective interventions are often limited by a lack of data concerning the impacts that overexploitation might have. To fill this gap, we tested the effects of overcollection in the genetic diversity and structure of Masdevallia rosea, a narrow distributed epiphytic orchid historically collected in Ecuador, in comparison with the widely distributed Pleurothallis lilijae. Genotyping based on AFLPs showed reduced levels of diversity in wild populations but most especially in the overcollected, M. rosea. Overall, genetic admixture was high in P. lilijae segregating populations by altitude levels while fewer genetic groups were found in M. rosea. Genetic differentiation was low in both species. A spatial genetic structure was found in P. lilijae depending on altitude levels, while no spatial genetic structure was found in M. rosea. These results suggest different scenarios for the two species: while gene flow over long distance is possible in P. lilijae, the same seems to be unlikely in M. rosea possibly due to the low levels of individuals in the known populations. In situ and ex situ conservation strategies should be applied to protect the genetic pool in these epiphytic orchid species, and to promote the connectivity between wild populations. Adopting measures to reduce overexploitation and to understand the impacts of harvesting in wild populations are necessary to strengthen the legal trade of orchids.

Denaturation of RNA secondary and tertiary structure by urea: simple unfolded state models and free energy parameters account for measured m-values.

To investigate the mechanism by which urea destabilizes RNA structure, urea-induced unfolding of four different RNA secondary and tertiary structures was quantified in terms of an m-value, the rate at which the free energy of unfolding changes with urea molality. From literature data and our osmometric study of a backbone analogue, we derived average interaction potentials (per square angstrom of solvent accessible surface) between urea and three kinds of RNA surfaces: phosphate, ribose, and base. Estimates of the increases in solvent accessible surface areas upon RNA denaturation were based on a simple model of unfolded RNA as a combination of helical and single-strand segments. These estimates, combined with the three interaction potentials and a term to account for interactions of urea with released ions, yield calculated m-values that are in good agreement with experimental values (200 mm monovalent salt). Agreement was obtained only if single-stranded RNAs were modeled in a highly stacked, A-form conformation. The primary driving force for urea-induced denaturation is the strong interaction of urea with the large surface areas of bases that become exposed upon denaturation of either RNA secondary or tertiary structure, though interactions of urea with backbone and released ions may account for up to a third of the m-value. Urea m-values for all four RNAs are salt-dependent, which we attribute to an increased extension (or decreased charge density) of unfolded RNAs with an increased urea concentration. The sensitivity of the urea m-value to base surface exposure makes it a potentially useful probe of the conformations of RNA unfolded states.