Sperm ultrastructure and spermatogenesis in the Japanese beetle Popillia japonica (Coleoptera, Scarabaeidae).

Popillia japonica is an invasive scarab beetle native to Japan that in 1916 invaded New Jersey in USA. From that moment onwards, the insect has spread invading several US states, Canada, the Azores, Italy and, recently, Switzerland. It is a severe agricultural pest included in the EU priority pest list being able to feed on more than 300 plant species and having an important biotic potential. The general morphology of the reproductive apparatus shows paired testes, each of them having six testicular lobes grouped in threes. From the ventral part of each testicular lobe, each containing about 20 follicles, an efferent vessel originates that fuses with the other efferent vessels to form the deferent duct. A pair of long tubular accessory glands is present. The deferent ducts and accessory glands fuse together into an ejaculatory duct before entering the aedeagus. The sperm is a typical pterygote sperm, 110 µm long, composed of a head and a tail. In the head a three-layered acrosome of about 6 µm in length and a nucleus of about 18 µm long are present. During sperm maturation two C-shaped structures appear in the cytoplasm from the opposite sides of the nucleus that then disappear in late spermatids. In the tail a typical 9 + 9 + 2 flagellar axoneme and two mitochondrial derivatives are present. Moreover, in the head-tail transition region the centriolar adjunct forms a sheath from which three elongated accessory bodies originate. Two of these accessory bodies are placed alongside the axoneme, whilst the third one is placed beneath the mitochondrial derivatives. Mature sperm are grouped in cysts containing about 256 sperm cells. A morphological comparison with related species is provided.

Adaptive management of invasive pests in natural protected areas: the case of Matsucoccus feytaudi in Central Italy.

Invasive species are a significant threat to affected ecosystems, having serious environmental, economic and social impacts. The maritime pine bast scale, Matsucoccus feytaudi Ducasse (Hemiptera: Matsucoccidae), causes serious damage to Pinus pinaster forests in SE France, Corsica and Italy where it has been introduced. This study illustrates the adaptive management plan implemented in the Migliarino, San Rossore, Massaciuccoli Regional Natural Park in Tuscany, Italy, where M. feytaudi arrived in 2004, leading to the decay of local P. pinaster stands. The management programme, aimed at slowing the establishment and growth of M. feytaudi, was carried out in the main sector of the park, Tenuta di San Rossore, to retard the destruction of the P. pinaster coastal strip protecting the more internal woodland from sea salt and to allow replacement of P. pinaster trees with a more stable broad-leaved wood. The combined use of mass trapping and silvicultural interventions, applied in a targeted manner according to distribution maps of pest captures and damage, helped to delay forest destruction compared with a nearby unmanaged area of the park Tenuta di Tombolo. Although M. feytaudi continued to spread during the management period, the populations remained at low levels for 6 years, showing a marked increase in 2012. During this period, the P. pinaster stands were reduced from 320 to 249 ha. The final result of this ongoing gradual conversion process will be transformation of the P. pinaster forest into Holm oak woods and Mediterranean shrub land, while P. pinaster will survive as clusters or blocks of trees.

Role of the quantiferon-TB test in ruling out pleural tuberculosis: a multi-centre study.

Diagnosing pleural tuberculosis (plTB) might be difficult due to limited sensitivity of conventional microbiology tools. As M. tuberculosis (MTB)-specific T cells are recruited into pleural space in plTB, their detection may provide useful clinical information. To this aim, in addition to standard diagnostic tests, we used the QuantiFERON-TB Gold In-Tube (QFT-IT) test in blood and pleural effusion (PE) samples from 48 patients with clinical suspicion of plTB, 18 (37.5%) of whom had confirmed plTB. Four of them (22.2%) tested positive with a nucleic acid amplification test for MTB. The tuberculin skin test was positive in most confirmed plTB cases (88.9%). Positive QFT-IT tests were significantly more frequent in patients with confirmed plTB, as compared to patients with an alternative diagnosis, both in blood (77.7 vs 36.6%, p=0.006) and in PE samples (83.3% vs 46.6%, p=0.02). In addition, both blood and PE MTB-stimulated IFN-gamma levels were significantly higher in plTB patients (p=0.03 and p=0.0049 vs non-plTB, respectively). In blood samples, QFT-IT had 77.8% sensitivity and 63.3% specificity, resulting in 56.0% positive (PPV) and 82.6% negative (NPV) predictive values. On PE, QFT-IT sensitivity was 83.3% and specificity 53.3% (PPV 51.7% and NPV 84.2%). The optimal AUC-derived cut-off for MTB-stimulated pleural IFN-gamma level was 3.01 IU/mL (77.8% sensitivity, 80% specificity, PPV 68.4% and NPV 82.8%). These data suggest that QFT-IT might have a role in ruling out plTB in clinical practice.

The effect of Tween 80 on eggshell permeabilization in Galleria mellonella (L.) (Lepidoptera, Pyralidae).

The development of a species-specific protocol for dechorionation and permeabilization of insect eggs is a necessary prerequisite to cryopreserve the embryos. Here we tested different procedures based on heptane or the surfactant Tween 80 as an alternative to alkane, evaluating their efficacy and toxicity on the early (24 h post-oviposition) and late (75 h post-oviposition) stage embryos. Heptane efficiently permeabilized the eggs of G. mellonella but the hatching rate ranged from 0.1 to 4.2 percent in the early stage and from 4.3 to 11.2 percent in the late stage. The embryos treated with 1.25 percent NaOCl + 0.08 percent Tween 80 for 2 min showed the same shrinkage and reswelling percentages as eggs exposed to heptane for 10 sec, with a significantly higher hatching percentage in the early (68.2 +/- 1.5 percent) and late stages (22.4 +/- 3.7 percent). Thus, 0.08 percent Tween 80 allows sufficient permeabilization of G. mellonella embryos without the high toxicity of alkane.

Interferon-gamma-release assays detect recent tuberculosis re-infection in elderly contacts.

The tuberculin skin test (TST) does not discriminate between recent and remote latent tuberculosis infection (LTBI). This study was carried out to test two interferon-gamma-based blood assays in recent contacts with high prevalence of remote LTBI. We performed a contact tracing investigation in a nursing home for the elderly, where elderly patients were exposed to a case of pulmonary tuberculosis. TST, QuantiFERON-TB Gold (QFT-G) and T-SPOT.TB (TS.TB) were performed 8 weeks after the end of potential exposure. IFN-gamma measurements were recorded and correlation with exposure was evaluated. Twenty-seven (37.5%), 32 (44.4%) and 16 (22.2%) subjects were TST, TS.TB and QFT-G positive, respectively; agreement between TS.TB and QFT-G was good among exposed subjects only (K=0.915, 0.218 in unexposed, p<0.001). When amounts of IFN-gamma were corrected for the number of producing T cells, specific IFN-gamma production was significantly different between exposed and unexposed individuals (16.75+/-5.40 vs 2.33+/-0.71 IFN-gamma IU/1000 SFC, p=0.0001). QFT-G and TS.TB provided discordant results among elderly contacts. Unlike TST, the specific IFN-gamma response might discriminate between recent and long-lasting tuberculosis infection.

Refinement of severely incomplete structures with maximum likelihood in BUSTER-TNT.

BUSTER-TNT is a maximum-likelihood macromolecular refinement package. BUSTER assembles the structural model, scales observed and calculated structure-factor amplitudes and computes the model likelihood, whilst TNT handles the stereochemistry and NCS restraints/constraints and shifts the atomic coordinates, B factors and occupancies. In real space, in addition to the traditional atomic and bulk-solvent models, BUSTER models the parts of the structure for which an atomic model is not yet available ('missing structure') as low-resolution probability distributions for the random positions of the missing atoms. In reciprocal space, the BUSTER structure-factor distribution in the complex plane is a two-dimensional Gaussian centred around the structure factor calculated from the atomic, bulk-solvent and missing-structure models. The errors associated with these three structural components are added to compute the overall spread of the Gaussian. When the atomic model is very incomplete, modelling of the missing structure and the consistency of the BUSTER statistical model help structure building and completion because (i) the accuracy of the overall scale factors is increased, (ii) the bias affecting atomic model refinement is reduced by accounting for some of the scattering from the missing structure, (iii) the addition of a spatial definition to the source of incompleteness improves on traditional Luzzati and sigmaA-based error models and (iv) the program can perform selective density modification in the regions of unbuilt structure alone.

The effect of long-term treatment with erdosteine on chronic obstructive pulmonary disease: the EQUALIFE Study.

Erdosteine is a new thiol compound with effects on bacterial adhesiveness as well as antioxidant and mucoactive properties. The EQUALIFE study, a fully randomized, double-blind, placebo-controlled, parallel-group, multicenter study, was designed to assets the effectiveness of long-term treatment with erdosteine in patients with moderate chronic obstructive pulmonary disease (COPD). One hundred and fifty-five patients received oral erdosteine, 300 mg b.i.d., or placebo for 8 months during the winter season to assess the effect of treatments on exacerbation rate, hospitalization, lung function and quality of life, assessed using the Short Form 36 and the St. George's Respiratory Questionnaire. A pharmacoeconomic analysis was also conducted to compare the two treatments. One hundred and twenty-four patients completed the study with erdosteine (n = 63) or placebo (n = 61). The group of COPD patients who received 8 months of continuous treatment with erdosteine had significantly fewer exacerbations and spent fewer days in the hospital than did the placebo group; furthermore, they had no loss of lung function. Patients in the erdosteine group also showed a significant improvement in health-related quality of life. The mean total COPD-related disease costs per patient were lower in the erdosteine group than in the placebo group over the study period. The results indicate that 8 months of treatment with erdosteine is effective in reducing exacerbation and hospitalization rates and in improving health status. The study suggests that erdosteine is likely to provide an important contribution to the therapy of patients with symptomatic COPD.

Complement regulation at the molecular level: the structure of decay-accelerating factor.

The human complement regulator CD55 is a key molecule protecting self-cells from complement-mediated lysis. X-ray diffraction and analytical ultracentrifugation data reveal a rod-like arrangement of four short consensus repeat (SCR) domains in both the crystal and solution. The stalk linking the four SCR domains to the glycosylphosphatidylinositol anchor is extended by the addition of 11 highly charged O-glycans and positions the domains an estimated 177 A above the membrane. Mutation mapping and hydrophobic potential analysis suggest that the interaction with the convertase, and thus complement regulation, depends on the burial of a hydrophobic patch centered on the linker between SCR domains 2 and 3.

Stereoselective synthesis of trifluoro- and monofluoro-analogues of frontalin and evaluation of their biological activity.

The stereoselective synthesis of both enantiomers of trifluoro frontalin (-)-(1S,5R)- and (+)-(1R,5S)-8, as well as of diastereomeric monofluoro frontalines (-)-(1R,2R,5R)-18 and (-)-(1R,2S,5R)-20, analogues of the bioactive component of the aggregation pheromone of the Scolytidae insect family, has been accomplished starting from (-)-(1R)- and (+)-(1S)-menthyl (S)-toluene-4-sulfinate as a source of chirality and methyl trifluoroacetate or fluoroacetate, respectively, as sources of fluorine. The C-1 stereocenters were installed via stereoselective epoxidation of beta-sulfinyl ketones 2 and 13 with diazomethane. The bicyclic core was obtained by totally stereocontrolled and chemoselective tandem Wacker oxidation/intramolecular ketalization of the intermediate unsatured sulfinyl diols 5, 15, and 19. Axially fluorinated (-)-20 elicited a strong electroantennographic response in laboratory tests on females of Dendroctonus micans, whereas equatorially fluorinated (-)-18 and the trifluoroanalogue (-)-8 showed modest responses. Field trials using (-)-20 were not indicative owing to the locally scarce population of D. micans, but it showed some attractiveness for other Coleoptera families.

High-resolution experimental phases for tryptophanyl-tRNA synthetase (TrpRS) complexed with tryptophanyl-5'AMP.

Native data, anomalous data at three wavelengths and an independent peak-wavelength data set for SeMet-substituted protein have been collected from cryoprotected crystals of the TrpRS-adenylate product (TAM) complex to a resolution limit of 1.7 A. Independent phase sets were developed using SHARP and improved by solvent flipping with SOLOMON using molecular envelopes derived from experimental densities for, respectively, peak-wavelength SAD data from four different crystals, MAD data and their M(S)IRAS combinations with native data. Hendrickson-Lattman phase-probability coefficients from each phase set were used in BUSTER to drive maximum-likelihood refinements of well defined parts of the previously refined room-temperature 2.9 A structure. Maximum-entropy completion followed by manual rebuilding was then used to generate a model for the missing segments, bound ligand and solvent molecules. Surprisingly, peak-wavelength SAD experiments produced the smallest phase errors relative to the refined structures. Selenomethionylated models deviate from one another by 0.25 A and from the native model by 0.38 A, but all have r.m.s. deviations of approximately 1.0 A from the 2.9 A model. Difference Fourier calculations between amplitudes from the 300 K experiment and the new amplitudes at 100 K using 1.7 A model phases show no significant structural changes arising from temperature variation or addition of cryoprotectant. The main differences between low- and high-resolution structures arise from correcting side-chain rotamers in the core of the protein as well as on the surface. These changes improve various structure-validation criteria.

Recovering experimental and theoretical electron densities in corundum using the multipolar model: IUCr Multipole Refinement Project.

This electron-density study on corundum (alpha-Al2O3) is part of the Multipole Refinement Project supported by the IUCr Commission on Charge, Spin and Momentum Densities. For this purpose, eight different data sets (two experimental and six theoretical) were chosen from which the electron density was derived by multipolar refinement (using the MOLLY program). The two experimental data sets were collected on a conventional CAD4 and at ESRF, ID11 with a CCD detector, respectively. The theoretical data sets consist of static, dynamic, static noisy and dynamic noisy moduli of structure factors calculated at the Hartree-Fock (HF) and density functional theory (DFT) levels. Comparisons of deformation and residual densities show that the multipolar analysis works satisfactorily but also indicate some drawbacks in the refinement. Some solutions and improvements during the refinements are proposed like contraction or expansion of the inner atomic shells or increasing the order of the spherical harmonic expansion.

In-house low-resolution X-ray crystallography.

It is demonstrated that standard in-house protein crystal X-ray diffraction apparatus can be used to measure very low resolution reflections with only a few modifications. The apparatus and modifications are described in detail and tested on two different macromolecular crystal samples: lysozyme and the 30S ribosomal subunit. Contrast-variation measurements on tetragonal hen egg-white lysozyme demonstrate the potential usefulness of the apparatus in providing accurate data for the determination of macromolecular envelopes. In contrast, the measurement of very low resolution diffraction from crystals of the 30S ribosome subunit illustrates how in-house facilities can provide data from small weakly diffracting crystals of a very large macromolecule.

Modelling prior distributions of atoms for macromolecular refinement and completion.

Until modelling is complete, macromolecular structures are refined in the absence of a model for some of the atoms in the crystal. Techniques for defining positional probability distributions of atoms, and using them to model the missing part of a macromolecular crystal structure and the bulk solvent, are described. The starting information may consist of either a tentative structural model for the missing atoms or an electron-density map. During structure completion and refinement, the use of probability distributions enables the retention of low-resolution phase information while avoiding premature commitment to uncertain higher resolution features. Homographic exponential modelling is proposed as a flexible, compact and robust parametrization that proves to be superior to a traditional Fourier expansion in approximating a model protein envelope. The homographic exponential model also has potential applications to ab initio phasing of Fourier amplitudes associated with macromolecular envelopes.