Diversity and Evolution of Entomocorticium (Russulales, Peniophoraceae), a Genus of Bark Beetle Mutualists Derived from Free-Living, Wood Rotting Peniophora.

Symbiosis between insects and fungi arose multiple times during the evolution of both groups, and some of the most biologically diverse and economically important are mutualisms in which the insects cultivate and feed on fungi. Among these are bark beetles, whose ascomycetous cultivars are better known and studied than their frequently-overlooked and poorly understood basidiomycetous partners. In this study, we propose five new species of Entomocorticium, fungal mutualists in the Russulales (Basidiomycota) that are mutualistic symbionts of scolytine beetles. We have isolated these fungi from the beetle mycangia, which are structures adapted for the selective storage and transportation of fungal mutualists. Herein, we present the most complete phylogeny of the closely related genera Entomocorticium and Peniophora and provide insights into how an insect-associated taxon (Entomocorticium) evolved from within a wood-decaying, wind-dispersed lineage (Peniophora). Our results indicate that following a transition from angiosperms to gymnosperms, fungal domestication by beetles facilitated the evolution and diversification of Entomocorticium. We additionally propose four new species: Entomocorticium fibulatum Araújo, Li & Hulcr, sp. nov.; E. belizense Araújo, Li & Hulcr, sp. nov.; E. perryae Araújo, Li & Hulcr, sp. nov.; and E. macrovesiculatum Araújo, Li, Six & Hulcr, sp. nov. Our findings highlight the fact that insect-fungi associations remain an understudied field and that these associations harbor a large reservoir of novel fungal species.

Impact of a Soil Conditioner Integrated into Fertilization Scheme on Orange and Lemon Seedling Physiological Performances.

Growers in Florida face unique challenges regarding maintaining proper citrus nutrition.Poor draining soils with low fertility, low C.E.C., and high rates of leaching are common in thisregion. In response to these challenges, interest has grown in products labeled as soil conditioners.Using a completely randomized experimental design, this greenhouse study tested the effects of 5different combinations of a traditional fertilizer (TF) and a new soil conditioner (SC) on lemon andorange seedling physiology. Eight-month-old 'Bearss' lemon and 'Valencia' sweet orange graftedon sour orange rootstocks were employed, and five repetitions were used for each treatment. Plantbiomass (dry weight), height, stem diameter, chlorophyll content, stomatal conductance andnutrient uptake were analyzed after 120 days of treatment. The results show that SC has a positiveimpact upon both chlorophyll levels and stomatal conductance values in both orange and lemonseedlings. However, based on dry weight growth data, we can only conclude that the SC waseffective for orange seedlings at 50% TF and 0.5% SC. Based on this short 120-day evaluation, theSC achieved positive growth promotion for orange (50% TF) but not for lemon seedlings.

Non-response bias in estimates of prevalence of club-based sport participation from an Australian national physical activity, recreation and sport survey.

BACKGROUND: An estimate of the prevalence of an activity derived from a sample survey is potentially subject to non-response bias, whereby people not involved in the activity are less likely to respond than those involved. Quantifying the extent of non-response bias is generally difficult, since it involves estimating differences between respondents for whom data is directly available from the survey, and non-respondents, for whom data is generally not directly or readily available. However, in the case of the Australian Exercise Recreation and Sport Survey (ERASS), comparative "gold standard" benchmarks exist for some aspects of the survey, in the form of state sporting association (SSA) registration databases, each of which purports to constitute a complete enumeration of club-based players of a particular sport. METHODS: ERASS estimates of the prevalence of participation in four major club-based team sports in the Australian state of Victoria in the year 2010 were compared with prevalences based on numbers of registered participants in the corresponding SSA databases. Comparisons were made for the adult population as a whole (ERASS scope being 15+ years of age), and for strata defined by age and geographical region. Because three of the four sports investigated are strongly sex-specific, no sex breakdowns were conducted. In each case the proportion of ERASS respondents reporting participation, with associated confidence limits, was compared with the corresponding SSA count expressed as a proportion of the population, to form an ERASS/SSA prevalence ratio with associated confidence limits. RESULTS: The 24 ERASS/SSA ratios ranged from 1.72 to 7.80. Most ratios lay in the range 2 to 3. The lower 95% confidence bound for the ratio was greater than 1.0 in 23 out of 24 cases. CONCLUSIONS: ERASS estimates of prevalence of these particular aspects of sport participation were higher than SSA estimates, to statistically significant degrees. The effect sizes (i.e. the discrepancies represented by the ratios) were large enough to be of great practical importance. It is conjectured that non-response bias is the most likely explanation for the discrepancies.

The influence of a Healthy Welcoming Environment on participation in club sport by adolescent girls: a longitudinal study.

BACKGROUND: This study investigated the perceived influence of a Healthy Welcoming Environment (HWE) on participation in sports clubs among adolescent girls, and how these perceptions changed longitudinally. HWE was defined in terms of a set of health promotion policies advocated by a health promotion foundation as the basis of sport club health promotion practice to promote structural reform in state sporting organisations and their affiliated associations and clubs. These included sports injury prevention, smoke-free, responsible serving of alcohol, sun protection, healthy eating, and welcoming and inclusive environments. METHODS: Year 7 and 11 female students from metropolitan (n = 17) and non-metropolitan secondary schools (n = 14) in Australia were invited to participate in three annual surveys. These surveys collected information about current or past membership of a sports club and the influence of HWEs on their decision to participate (or not) in a sports club. RESULTS: Year 7 (n = 328; 74.5%) and Year 11 (n = 112; 25.5%) female students completed all three waves (19.6% response rate; 82.7 and 74.0% retention rate). Most agreed that characteristics of HWEs were a positive influence on their participation in sports clubs, except those relating to alcohol and Sunsmart. Welcoming factors had consistent high agreement among respondents. Alcohol and friendliness factors of the club were regarded as being positively influential by higher percentages of non-metropolitan than metropolitan respondents. CONCLUSIONS: Welcoming factors were the most positive influences on decisions to participate in sports clubs. These factors may be important in reducing barriers to sport participation. Strategies supporting the social environment within sports clubs should be prioritised.

Combining Design and Selection to Create Novel Protein-Peptide Interactions.

The ability to design new protein-protein interactions (PPIs) has many applications in biotechnology and medicine. The goal of designed PPIs is to achieve both high affinity and specificity for the target protein. A great challenge in protein design is to identify such proteins from an enormous number of potential sequences. Many computational and experimental methods have been developed to contend with this challenge. Here we describe one particularly powerful approach-semirational design-that combines design and selection. This approach has been applied to generate new PPIs for many applications, including novel affinity reagents for protein detection/purification and bioorthogonal modules for synthetic biology (Jackrel, Valverde, & Regan, 2009; Sawyer et al., 2014; Speltz, Brown, Hajare, Schlieker, & Regan, 2015; Speltz, Nathan, & Regan, 2015).

Scn1a dysfunction alters behavior but not the effect of stress on seizure response.

Mutations in the voltage-gated sodium channel gene SCN1A are responsible for a number of epilepsy disorders, including genetic epilepsy with febrile seizures plus (GEFS+) and Dravet syndrome. In addition, dysfunction in SCN1A is increasingly being linked to neuropsychiatric abnormalities, social deficits and cognitive disabilities. We have previously reported that mice heterozygous for the SCN1A R1648H mutation identified in a GEFS+ family have infrequent spontaneous seizures, increased susceptibility to chemically and hyperthermia-induced generalized seizures and sleep abnormalities. In this study, we characterized the behavior of heterozygous mice expressing the SCN1A R1648H mutation (Scn1a(RH/+)) and the effect of stress on spontaneous and induced seizures. We also examined the effect of the R1648H mutation on the hypothalamic-pituitary-adrenal (HPA) axis response. We confirmed our previous finding that Scn1a(RH/+) mutants are hyperactive, and also identified deficits in social behavior, spatial memory, cued fear conditioning, pre-pulse inhibition and risk assessment. Furthermore, while exposure to a stressor did increase seizure susceptibility, the effect seen in the Scn1a(RH/+) mutants was similar to that seen in wild-type littermates. In addition, Scn1a dysfunction does not appear to alter HPA axis function in adult animals. Our results suggest that the behavioral abnormalities associated with Scn1a dysfunction encompass a wider range of phenotypes than previously reported and factors such as stress exposure may alter disease severity in patients with SCN1A mutations.

Tracking large solid constructs suspended in a rotating bioreactor: A combined experimental and theoretical study.

We present a combined experimental and theoretical study of the trajectory of a large solid cylindrical disc suspended within a fluid-filled rotating cylindrical vessel. The experimental set-up is relevant to tissue-engineering applications where a disc-shaped porous scaffold is seeded with cells to be cultured, placed within a bioreactor filled with nutrient-rich culture medium, which is then rotated in a vertical plane to keep the growing tissue construct suspended in a state of "free fall." The experimental results are compared with theoretical predictions based on the model of Cummings and Waters (2007), who showed that the suspended disc executes a periodic motion. For anticlockwise vessel rotation three regimes were identified: (i) disc remains suspended at a fixed position on the right-hand side of the bioreactor; (ii) disc executes a periodic oscillatory motion on the right-hand side of the bioreactor; and (iii) disc orbits the bioreactor. All three regimes are captured experimentally, and good agreement between theory and experiment is obtained. For the tissue engineering application, computation of the fluid dynamics allows the nutrient concentration field surrounding a tissue construct (a property that cannot be measured experimentally) to be determined (Cummings and Waters, 2007). The implications for experimental cell-culture protocols are discussed.

In situ monitoring of 3D in vitro cell aggregation using an optical imaging system.

Bioreactor systems that maintain cells and tissues in suspension are increasingly popular for culturing 3D constructs to avoid the loss of in vivo cell function associated with traditional 2D culture methods. There is a need for the online monitoring of such systems to provide better understanding and control of the processes involved and to prevent the disruption of these processes caused by offline sampling and endpoint analysis. We describe a system for the imaging and analysis of cell aggregation, over long periods, within a high aspect rotating vessel (HARV). The system exploits side illumination, using an adjustable beam pattern, to restrict the detected light to that scattered by the cell aggregates, thus eliminating the need for the fluorescent labeling of the cells. The in situ aggregation of mammalian cells (MCF-7 breast carcinoma cells) was monitored over an 8 h period and image sequences showing the growth and motion of the aggregates within the bioreactor were obtained. Detailed size and population data have been derived characterizing the development of the aggregates during this time. We show how the number of resolvable aggregates increases to reach a peak and then declines as these aggregates merge. Once formed, remaining aggregates are found to consolidate to form more tightly packed bodies, typically reducing in cross-sectional area by one third. These results provide the basis for the development of an automated feedback system to control the growth of 3D cell cultures for repeatable, reliable, and quality controlled experimentation.

Denaturing high performance liquid chromatography: high throughput mutation screening in familial hypertrophic cardiomyopathy and SNP genotyping in motor neurone disease.

AIMS: To evaluate the usefulness of denaturing high performance liquid chromatography (DHPLC) as a high throughput tool in: (1) DNA mutation detection in familial hypertrophic cardiomyopathy (FHC), and (2) single nucleotide polymorphism (SNP) discovery and validation in sporadic motor neurone disease (MND). METHODS: The coding sequence and intron-exon boundaries of the cardiac beta myosin heavy chain gene (MYH7) were screened by DHPLC for mutation identification in 150 unrelated patients diagnosed with FHC. One hundred and forty patients with sporadic MND were genotyped for the A67T SNP in the poliovirus receptor gene. All DHPLC positive signals were confirmed by conventional methods. RESULTS: Mutation screening of MYH7 covered 10 kb with a total of 5700 amplicons, and more than 6750 DHPLC injections were completed within 35 days. The causative mutation was identified in 14% of FHC cases, including seven novel missense mutations (L227V, E328G, K351E, V411I, M435T, E894G, and E927K). Genotyping of the A67T SNP was performed at two different temperatures both in MND cases and 280 controls. This coding SNP was found more frequently in MND cases (13.6%) than in controls (6.8%). Furthermore, 19 and two SNPs were identified in MYH7 and the poliovirus receptor gene, respectively, during DHPLC screening. CONCLUSIONS: DHPLC is a high throughput, sensitive, specific, and robust platform for the detection of DNA variants, such as disease causing mutations or SNPs. It enables rapid and accurate screening of large genomic regions.

Structure-activity relationship of biaryl acylsulfonamide analogues on the human EP(3) prostanoid receptor.

Potent and selective ligands for the human EP3 prostanoid receptor are described. Biaryl compounds bearing a tethered ortho substituted acidic moiety were identified as potent EP3 antagonists based on the SAR described herein. The binding affinity of key compounds on all eight human prostanoid receptors is reported.

Selective modulation of chemokinesis, degranulation, and apoptosis in eosinophils through the PGD2 receptors CRTH2 and DP.

BACKGROUND: PGD(2) is the major prostanoid released by mast cells during an allergic response. Its role in the allergic response, however, remains unclear. OBJECTIVE: Because the accumulation of eosinophils is a feature of allergic reactions, we investigated the role of PGD(2) in the modulation of eosinophil function. METHODS: Circulating human eosinophils were isolated and challenged with PGD(2). The effects of PGD(2) on various eosinophil functions were then analyzed. RESULTS: PGD(2) binds with high affinity preferentially to 2 receptors, DP and chemoattractant receptor-homologous molecule expressed on T(H)2 cells (CRTH2). We show that both DP and CRTH2 are detectable on circulating eosinophils. We demonstrate that PGD(2) (1-10 nmol/L) induces a rapid change in human eosinophil morphology and an increase in chemokinesis and promotes eosinophil degranulation. These effects are induced by the CRTH2-selective agonist 13-14-dihydro-15-keto-PGD(2) (DK-PGD(2)) but not by the DP-selective agonist BW245C. These results suggest a role for CRTH2 in the modulation of eosinophil movement and in triggering the release of cytotoxic proteins. Finally, we demonstrate that BW245C, but not DK-PGD(2), can delay the onset of apoptosis in cultured eosinophils, presumably through interaction with DP. CONCLUSION: These data support the hypothesis that PGD(2) controls eosinophil functions through 2 pharmacologically distinct receptors with independent functions. Blockade of PGD(2)-mediated effects on human eosinophils may reduce the damage caused by these cells during an allergic response, but inhibition of both receptors may be required.

Molecular cloning and functional characterization of murine cysteinyl-leukotriene 1 (CysLT(1)) receptors.

We sought to clone and characterize the murine cysteinyl-leukotriene D(4) receptor (mCysLT(1)R) to complement our studies with leukotriene-deficient mice. A cDNA, cloned from trachea mRNA by reverse transcriptase-polymerase chain reaction, has two potential initiator ATG codons that would encode for polypeptides of 352 and 339 amino acids, respectively. These two potential forms, predicted to be seven transmembrane-spanning domain proteins, have 87% amino acid identity with the human CysLT(1) receptor (hCysLT(1)R). Membrane fractions of Cos-7 cells transiently expressing the short mCysLT(1)R demonstrated high affinity and specific binding for leukotriene D(4) (LTD(4), K(d) = 0.25 +/- 0.04 nM). In competition binding experiments, LTD(4) was the most potent competitor (K(i) = 0.8 +/- 0.2 nM) followed by LTE(4) and LTC(4) (K(i) = 86.6 +/- 24.5 and 100.1 +/- 17.1 nM, respectively) and LTB(4) (K(i) > 1.5 microM). Binding of LTD(4) was competitively inhibited by the specific CysLT(1) receptor antagonists MK-571 [(+)-3-(((3-(2-(7-chloro-2-quinolinyl)ethenyl)phenyl) ((3-(dimethylamino)-3-oxopropyl)thio)methyl)thio)propanoic acid], pranlukast (Onon), and zafirlukast (Accolate), while the CysLT(1)/CysLT(2) receptor antagonist BAY-u9773 [6(R)-(4'-carboxyphenylthio)-5(S)-hydroxy-7(E),9(E),11(Z),14(Z)-eicosatetrenoic acid] was 1000 times less potent than LTD(4). In transiently transfected HEK293-T cells expressing either the long or short form of mCysLT(1)R, LTD(4) induced an increase of intracellular calcium. In Xenopus laevis melanophores transiently expressing either isoform, LTD(4) induced the dispersion of pigment granules, consistent with the activation by LTD(4) of a G(alphaq) (calcium) pathway. Functional elucidation of mCysLT(1)R properties as described here will enable further experiments to clarify the selective role of LTD(4) in murine models of inflammation and asthma.

Structure-activity relationship of cinnamic acylsulfonamide analogues on the human EP3 prostanoid receptor.

Potent and selective antagonists of the human EP3 receptor have been identified. The structure-activity relationship of the chemical series was conducted and we found several analogues displaying sub-nanomolar K(i) values at the EP3 receptor and micromolar activities at the EP1, EP2 and EP4 receptors. The effect of added human serum albumin (HSA) on the binding affinity at the EP3 receptor was also investigated.

Prostaglandin receptor EP(4) mediates the bone anabolic effects of PGE(2).

Prostaglandin (PG) E(2) is a potent inducer of cortical and trabecular bone formation in humans and animals. Although the bone anabolic action of PGE(2) is well documented, the cellular and molecular mechanisms that mediate this effect remain unclear. This study was undertaken to examine the effect of pharmacological inactivation of the prostanoid receptor EP(4), one of the PGE(2) receptors, on PGE(2)-induced bone formation in vivo. We first determined the ability of EP(4)A, an EP(4)-selective ligand, to act as an antagonist. PGE(2) increases intracellular cAMP and suppresses apoptosis in the RP-1 periosteal cell line. Both effects were reversed by EP(4)A, suggesting that EP(4)A acts as an EP(4) antagonist in the cells at concentrations consistent with its in vitro binding to EP(4). We then examined the effect of EP(4) on bone formation induced by PGE(2) in young rats. Five- to 6-week-old rats were treated with PGE(2) (6 mg/kg/day) in the presence or absence of EP(4)A (10 mg/kg/day) for 12 days. We found that treatment with EP(4)A suppresses the increase in trabecular bone volume induced by PGE(2). This effect is accompanied by a suppression of bone formation indices: serum osteocalcin, extent of labeled surface, and extent of trabecular number, suggesting that the reduction in bone volume is due most likely to decreased bone formation. The pharmacological evidence presented here provides strong support for the hypothesis that the bone anabolic effect of PGE(2) in rats is mediated by the EP(4) receptor.

Key structural features of prostaglandin E(2) and prostanoid analogs involved in binding and activation of the human EP(1) prostanoid receptor.

The structure-activity relationship (SAR) of prostaglandin (PG) E(2) at the human EP(1) prostanoid receptor (designated hEP(1)) was examined via the binding and activation of this receptor by a series of 55 prostanoids and analogs. Using clonal human embryonic kidney 293 cell lines expressing recombinant hEP(1), affinity (K(i)), potency (EC(50)), and efficacy data were obtained using a radioligand competitive binding assay and an aequorin-based calcium functional assay. All compounds behaved as full agonists (90-100% of the response elicited by PGE(2)) in this assay, and the correlation between the K(i) and EC(50) values was highly significant (R(2) = 0.86). The results from the SAR analysis can be summarized as follows: 1) the existence and configuration of hydroxyl groups at the 11 and 15 positions of PGE(2) and prostanoid analog structures play a critical role in agonist activity; 2) the carboxyl group is also important for activity and modification of the carboxylic acid to various esters results in greatly reduced affinity and potency; 3) the activity of structures with moderate or weak potency can be enhanced by modification of the omega-tail; and 4) modifications to the ketone at the 9-position are better tolerated, with 9-deoxy-9-methylene-PGE(2) being the most potent agonist tested in the functional assay. The impact of other modifications on agonist potency is also discussed. The results from this study have identified, for the first time, the key structural features of PGE(2) and related prostanoids and prostanoid analogs necessary for activation of hEP(1).

Structure-activity relationship on the human EP3 prostanoid receptor by use of solid-support chemistry.

Potent and selective EP3 receptor ligands were found by making a library using solid-support chemistry. These compounds can be obtained by a Suzuki coupling reaction of a solid-supported benzyl bromide using various boronic acids. The yields obtained for this reaction were in the range of 24-95% of arylmethyl cinnamic acid 1 after cleavage from the Wang resin.

Placental anomalies in a viable cloned calf.

Placental anomalies are associated with a high mortality rate in mammalian cloning programs. In this report, we detail the very unusual occurrence of a grossly abnormal placenta that supported a viable cloned calf to term. The placenta was recovered intact 3 h following birth, and its weight was within normal limits (4.3 kg). The chorioallantois of the cloned transgenic female Holstein calf contained only 26 cotyledons. Twelve of these were enlarged and functional. Six were poorly developed, and eight were degenerating. The 12 functional cotyledons ranged in diameter from 8 to 20 cm. The nongravid horn had six rudimentary (<5 cm in diameter) cotyledons and eight cotyledons that remained as oval, mineralized plaques. Despite the reduction in number of placentomes, there was no adventitial placentation. Although this report documents observations from a single case, it does show that a morphologically deficient placenta was able to support development to term and resulted in a viable calf.

Species-specific monoclonal antibodies to Escherichia coli-expressed p36 cytosolic protein of Mycoplasma hyopneumoniae.

The p36 protein of Mycoplasma hyopneumoniae is a cytosolic protein carrying species-specific antigenic determinants. Based on the genomic sequence of the reference strain ATCC 25934, primers were designed for PCR amplification of the p36-encoding gene (948 bp). These primers were shown to be specific to M. hyopneumoniae since no DNA amplicons could be obtained with other mycoplasma species and pathogenic bacteria that commonly colonize the porcine respiratory tract. The amplified p36 gene was subcloned into the pGEX-4T-1 vector to be expressed in Escherichia coli as a fusion protein with glutathione S-transferase (GST). The GST-p36 recombinant fusion protein was purified by affinity chromatography and cut by thrombin, and the enriched p36 protein was used to immunize female BALB/c mice for the production of anti-p36 monoclonal antibodies (MAbs). The polypeptide specificity of the nine MAbs obtained was confirmed by Western immunoblotting with cell lysates prepared from the homologous strain. Cross-reactivity studies of the anti-p36 MAbs towards two other M. hyopneumoniae reference strains (ATCC 25095 and J strains) and Quebec field strains that had been isolated in culture suggested that these anti-p36 MAbs were directed against a highly conserved epitope, or closely located epitopes, of the p36 protein. No reactivity was demonstrated against other mycoplasma species tested. Clinical signs and lesions suggestive of enzootic pneumonia were reproduced in specific-pathogen-free pigs infected experimentally with a virulent Quebec field strain (IAF-DM9827) of M. hyopneumoniae. The bacteria could be recovered from lung homogenates of pigs that were killed after the 3-week observation period by both PCR and cultivation procedures. Furthermore, the anti-p36 MAbs permitted effective detection by indirect immunofluorescence of M. hyopneumoniae in frozen lung sections from experimentally infected pigs. However, attempts to use the recombinant p36 protein as an antigen in an indirect enzyme-linked immunosorbent assay for the detection of antibodies in sera from convalescent pigs showed no correlation with clinical and pathological findings.