Do Insectivorous Birds use Volatile Organic Compounds from Plants as Olfactory Foraging Cues? Three Experimental Tests

Some insectivorous birds orient towards insect‐defoliated trees even when they do not see the foliar damage or the herbivores. There are, however, only a few studies that have examined the mechanisms behind this foraging behaviour. Previous studies suggest that birds can use olfactory foraging cues (e.g. volatile organic compounds (VOCs) emitted by defoliated plants), indirect visual cues or a combination of the two sensory cues. VOCs from insect‐defoliated plants are known to attract natural enemies of herbivores, and researchers have hypothesized that VOCs could also act as olfactory foraging cues for birds. We conducted three experiments across a range of spatial scales to test this hypothesis. In each experiment, birds were presented with olfactory cues and their behavioural responses or foraging outcomes were observed. In the first experiment, two different VOC blends, designed to simulate the volatile emissions of mountain birch (Betula pubescens ssp. czerepanovii) after defoliation by autumnal moth (Epirrita autumnata) larvae, were used in behavioural experiments in aviaries with pied flycatchers (Ficedula hypoleuca). The second experiment was a field‐based trial of bird foraging efficiency; the same VOC blends were applied to mountain birches, silver birches (B. pendula) and European white birches (B. pubescens) with plasticine larvae attached to the trees to serve as artificial prey for birds and provide a means to monitor predation rate. In the third experiment, the attractiveness of silver birch saplings defoliated by autumnal moth larvae versus intact controls was tested with great tits (Parus major) and blue tits (Cyanistes caeruleus) in an aviary. Birds did not orient towards either artificial or real trees with VOC supplements or towards herbivore‐damaged saplings when these saplings and undamaged alternatives were hidden from view. These findings do not support the hypothesis that olfactory foraging cues are necessary in the attraction of birds to herbivore‐damaged trees.     

SH3 domain-mediated recruitment of host cell amphiphysins by alphavirus nsP3 promotes viral RNA replication

Among the four non-structural proteins of alphaviruses the function of nsP3 is the least well understood. NsP3 is a component of the viral replication complex, and composed of a conserved aminoterminal macro domain implicated in viral RNA synthesis, and a poorly conserved carboxyterminal region. Despite the lack of overall homology we noted a carboxyterminal proline-rich sequence motif shared by many alphaviral nsP3 proteins, and found it to serve as a preferred target site for the Src-homology 3 (SH3) domains of amphiphysin-1 and -2. Nsp3 proteins of Semliki Forest (SFV), Sindbis (SINV), and Chikungunya viruses all showed avid and SH3-dependent binding to amphiphysins. Upon alphavirus infection the intracellular distribution of amphiphysin was dramatically altered and colocalized with nsP3. Mutations in nsP3 disrupting the amphiphysin SH3 binding motif as well as RNAi-mediated silencing of amphiphysin-2 expression resulted in impaired viral RNA replication in HeLa cells infected with SINV or SFV. Infection of Balb/c mice with SFV carrying an SH3 binding-defective nsP3 was associated with significantly decreased mortality. These data establish SH3 domain-mediated binding of nsP3 with amphiphysin as an important host cell interaction promoting alphavirus replication.     

Biochemical and structural insights into the mechanisms of SARS coronavirus RNA ribose 2'-O-methylation by nsp16/nsp10 protein complex

The 5'-cap structure is a distinct feature of eukaryotic mRNAs, and eukaryotic viruses generally modify the 5'-end of viral RNAs to mimic cellular mRNA structure, which is important for RNA stability, protein translation and viral immune escape. SARS coronavirus (SARS-CoV) encodes two S-adenosyl-L-methionine (SAM)-dependent methyltransferases (MTase) which sequentially methylate the RNA cap at guanosine-N7 and ribose 2'-O positions, catalyzed by nsp14 N7-MTase and nsp16 2'-O-MTase, respectively. A unique feature for SARS-CoV is that nsp16 requires non-structural protein nsp10 as a stimulatory factor to execute its MTase activity. Here we report the biochemical characterization of SARS-CoV 2'-O-MTase and the crystal structure of nsp16/nsp10 complex bound with methyl donor SAM. We found that SARS-CoV nsp16 MTase methylated m7GpppA-RNA but not m7GpppG-RNA, which is in contrast with nsp14 MTase that functions in a sequence-independent manner. We demonstrated that nsp10 is required for nsp16 to bind both m7GpppA-RNA substrate and SAM cofactor. Structural analysis revealed that nsp16 possesses the canonical scaffold of MTase and associates with nsp10 at 1∶1 ratio. The structure of the nsp16/nsp10 interaction interface shows that nsp10 may stabilize the SAM-binding pocket and extend the substrate RNA-binding groove of nsp16, consistent with the findings in biochemical assays. These results suggest that nsp16/nsp10 interface may represent a better drug target than the viral MTase active site for developing highly specific anti-coronavirus drugs.     

Simple and inexpensive immunoassay-based diagnostic tests

Simple and inexpensive yet sensitive and robust diagnostic tests are critically needed for resource-poor settings to enable timely diagnosis and effective use of limited health care resources. Current tests are often too expensive, too slow, or have compromised clinical performance, and they often require health care professional to perform the test. In addition, most assays are not intended to be used in extreme environmental conditions, but their performance may be affected by high temperatures and humidity often encountered in resource-poor settings. This review provides an overview of current immunoassay technologies and their advantages and limitations with respect to their feasibility to resource-poor settings. Future trends of immunoassay development for decentralized testing are also discussed. Homogeneous assays as such are out of the scope of this article because they are generally not yet sensitive enough or otherwise less feasible for inexpensive rapid diagnostic tests.     

Inhibitors of alphavirus entry and replication identified with a stable Chikungunya replicon cell line and virus-based assays

Chikungunya virus (CHIKV), an alphavirus, has recently caused epidemic outbreaks and is therefore considered a re-emerging pathogen for which no effective treatment is available. In this study, a CHIKV replicon containing the virus replicase proteins together with puromycin acetyltransferase, EGFP and Renilla luciferase marker genes was constructed. The replicon was transfected into BHK cells to yield a stable cell line. A non-cytopathic phenotype was achieved by a Pro718 to Gly substitution and a five amino acid insertion within non-structural protein 2 (nsP2), obtained through selection for stable growth. Characterization of the replicon cell line by Northern blotting analysis revealed reduced levels of viral RNA synthesis. The CHIKV replicon cell line was validated for antiviral screening in 96-well format and used for a focused screen of 356 compounds (natural compounds and clinically approved drugs). The 5,7-dihydroxyflavones apigenin, chrysin, naringenin and silybin were found to suppress activities of EGFP and Rluc marker genes expressed by the CHIKV replicon. In a concomitant screen against Semliki Forest virus (SFV), their anti-alphaviral activity was confirmed and several additional inhibitors of SFV with IC₅₀ values between 0.4 and 24 µM were identified. Chlorpromazine and five other compounds with a 10H-phenothiazinyl structure were shown to inhibit SFV entry using a novel entry assay based on a temperature-sensitive SFV mutant. These compounds also reduced SFV and Sindbis virus-induced cytopathic effect and inhibited SFV virion production in virus yield experiments. Finally, antiviral effects of selected compounds were confirmed using infectious CHIKV. In summary, the presented approach for discovering alphaviral inhibitors enabled us to identify potential lead structures for the development of alphavirus entry and replication phase inhibitors as well as demonstrated the usefulness of CHIKV replicon and SFV as biosafe surrogate models for anti-CHIKV screening.     

Local genetic population structure in an endangered plant species, Silene tatarica (Caryophyllaceae)

Genetic substructuring in plant populations may evolve as a consequence of sampling events that occur when the population is founded or regenerated, or if gene dispersal by pollen and seeds is restricted within a population. Silene tatarica is an endangered, perennial plant species growing along periodically disturbed riverbanks in northern Finland. We investigated the mechanism behind the microspatial genetic structure of S. tatarica in four subpopulations using amplified fragment length polymorphism markers. Spatial autocorrelation revealed clear spatial genetic structure in each subpopulation, even though the pattern diminished in older subpopulations. Parentage analysis in an isolated island subpopulation indicated a very low level of selfing and avoidance of breeding between close relatives. The mean estimated pollen dispersal distance (24.10 m; SD = 10.5) was significantly longer and the mean seed dispersal distance (9.07 m; SD = 9.23) was considerably shorter than the mean distance between the individuals (19.20 m; SD = 13.80). The estimated indirect and direct estimates of neighbourhood sizes in this subpopulation were very similar, 32.1 and 37.6, respectively. Our results suggested that the local spatial genetic structure in S. tatarica was attributed merely to the isolation-by-distance process rather than founder effect, and despite free pollen movement across population, restricted seed dispersal maintains local genetic structure in this species.     

Medroxyprogesterone improves nocturnal breathing in postmenopausal women with chronic obstructive pulmonary disease

Background

Progestins as respiratory stimulants in chronic obstructive pulmonary disease (COPD) have been investigated in males and during wakefulness. However, sleep and gender may influence therapeutic responses. We investigated the effects of a 2-week medroxyprogesterone acetate (MPA) therapy on sleep and nocturnal breathing in postmenopausal women.

Methods

A single-blind placebo-controlled trial was performed in 15 postmenopausal women with moderate to severe COPD. A 12-week trial included 2-week treatment periods with placebo and MPA (60 mg/d/14 days). All patients underwent a polysomnography with monitoring of SaO₂ and transcutaneous PCO₂ (tcCO₂) at baseline, with placebo, with medroxyprogesterone acetate (MPA 60 mg/d/14 days), and three and six weeks after cessation of MPA.

Results

Thirteen patients completed the trial. At baseline, the average ± SD of SaO₂ mean was 90.6 ± 3.2 % and the median of SaO₂ nadir 84.8 % (interquartile range, IQR 6.1). MPA improved them by 1.7 ± 1.6 %-units (95 % confidence interval (CI) 0.56, 2.8) and by 3.9 %-units (IQR 4.9; 95% CI 0.24, 10.2), respectively. The average of tcCO₂ median was 6.0 ± 0.9 kPa and decreased with MPA by 0.9 ± 0.5 kPa (95% CI -1.3, -0.54). MPA improved SaO₂ nadir and tcCO₂ median also during REM sleep. Three weeks after cessation of MPA, the SaO₂ mean remained 1.4 ± 1.8 %-units higher than at baseline, the difference being not significant (95% CI -0.03, 2.8). SaO₂ nadir was 2.7 %-units (IQR 4.9; 95% CI 0.06, 18.7) higher than at baseline. Increases in SaO₂ mean and SaO₂ nadir during sleep with MPA were inversely associated with baseline SaO₂ mean (r = -0.70, p = 0.032) and baseline SaO₂ nadir (r = -0.77, p = 0.008), respectively. Treatment response in SaO₂ mean, SaO₂ nadir and tcCO₂ levels did not associate with pack-years smoked, age, BMI, spirometric results or sleep variables.

Conclusion

MPA-induced respiratory improvement in postmenopausal women seems to be consistent and prolonged. The improvement was greater in patients with lower baseline SaO₂ values. Long-term studies in females are warranted.     

Characterization of recombinant amino-terminal NC4 domain of human collagen IX: interaction with glycosaminoglycans and cartilage oligomeric matrix protein

The N-terminal NC4 domain of collagen IX is a globular structure projecting away from the surface of the cartilage collagen fibril. Several interactions have been suggested for this domain, reflecting its location and its characteristic high isoelectric point. In an attempt to characterize the NC4 domain in more detail, we set up a prokaryotic expression system to produce the domain. The purified 27.5-kDa product was analyzed for its glycosaminoglycan-binding potential by surface plasmon resonance and solid-state assays. The results show that the NC4 domain of collagen IX specifically binds heparin with a K(d) of 0.6 microm, and the full-length recombinant collagen IX has an even stronger interaction with heparin, with an apparent K(d) of 3.6 nm. The heparin-binding site of the NC4 domain was located in the extreme N terminus, containing a heparin-binding consensus sequence, whereas electron microscopy suggested the presence of at least three additional heparin-binding sites on full-length collagen IX. The NC4 domain was also shown to bind cartilage oligomeric matrix protein. This interaction and the association of cartilage oligomeric matrix protein with other regions of collagen IX were found to be heparin-competitive. Circular dichroism analyses of the NC4 domain indicated the presence of stabilizing disulfide bonds and a thermal denaturation point of about 80 degrees C. The pattern of disulfide bond formation within the NC4 domain was identified by tryptic peptide mass mapping of the NC4 in native and reduced states. A similar pattern was demonstrated for the NC4 domain of full-length recombinant collagen IX.     

Nε-Modified lysine containing inhibitors for SIRT1 and SIRT2

Sirtuins catalyze the NAD⁺ dependent deacetylation of N^ε-acetyl lysine residues to nicotinamide, O′-acetyl-ADP-ribose (OAADPR) and N^ε-deacetylated lysine. Here, an easy-to-synthesize Ac-Ala-Lys-Ala sequence has been used as a probe for the screening of novel N^ε-modified lysine containing inhibitors against SIRT1 and SIRT2. N^ε-Selenoacetyl and N^ε-isothiovaleryl were the most potent moieties found in this study, comparable to the widely studied N^ε-thioacetyl group. The N^ε-3,3-dimethylacryl and N^ε-isovaleryl moieties gave significant inhibition in comparison to the N^ε-acetyl group present in the substrates. In addition, the studied N^ε-alkanoyl, N^ε-α,β-unsaturated carbonyl and N^ε-aroyl moieties showed that the acetyl binding pocket can accept rather large groups, but is sensitive to even small changes in electronic and steric properties of the N^ε-modification. These results are applicable for further screening of N^ε-acetyl analogues.